Engineered imine reductases and methods for the reductive amination of ketone and amine compounds

ABSTRACT

The present disclosure provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.

1. CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of co-pending U.S. patentapplication Ser. No. 16/054,843, filed on Aug. 3, 2018, which isContinuation of U.S. patent application Ser. No. 15/899,834, filed onFeb. 20, 2018, now U.S. Pat. No. 10,066,250, which is a Continuation ofU.S. patent application Ser. No. 15/792,446, filed Oct. 24, 2017, nowU.S. Pat. No. 9,932,613, which is a Continuation of U.S. patentapplication Ser. No. 15/710,462, filed Sep. 20, 2017, now U.S. Pat. No.9,828,614, which is a Continuation of U.S. patent application Ser. No.15/605,061, filed May 25, 2017, now U.S. Pat. No. 9,803,224, which is aContinuation of U.S. patent application Ser. No. 15/286,900, filed Oct.6, 2016, now U.S. Pat. No. 9,695,451, which is a Continuation of U.S.patent application Ser. No. 15/048,887, filed Feb. 19, 2016, now U.S.Pat. No. 9,487,760, which is a Continuation of U.S. patent applicationSer. No. 14/887,943, filed Oct. 20, 2015, now U.S. Pat. No. 9,296,993,which is a Divisional of U.S. patent application Ser. No. 13/890,944,filed May 9, 2013, now U.S. Pat. No. 9,193,957, which claims benefitunder 35 U.S.C. § 119(e) of U.S. Pat. Appln. Ser. No. 61/646,100, filedMay 11, 2012, the contents of all of which are incorporated herein byreference.

2. TECHNICAL FIELD

The disclosure relates to engineered polypeptides having imine reductaseactivity useful for the conversion of various ketone and aminesubstrates to secondary and tertiary amine products.

3. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-120US2_ST25.txt”, a creation date of May 8, 2013, anda size of 1,729,414 bytes. The Sequence Listing filed via EFS-Web ispart of the specification and is incorporated in its entirety byreference herein.

4. BACKGROUND

Chiral secondary and tertiary amines are important building blocks inthe pharmaceutical industry. There are no efficient biocatalytic routesknown to produce this class of chiral amine compounds. The existingchemical methods use chiral boron reagents or multi step synthesis.

There are a few reports in the literature of the biocatalytic synthesisof secondary amines. Whole cells of the anaerobic bacteriumAcetobacterium woodii imine reductase activity was reported to reducebenzylidine imines and butylidine imines (Chadha, et al., 2008,Tetrahedron:Asymmetry. 19: 93-96). Another report uses benzaldehyde orbutyraldehyde and butyl amine or aniline in aqueous medium using wholecells of Acetobacterium woodii (Stephens et al., 2004, Tetrahedron.60:753-758). Streptomyces sp. GF3587 and GF3546 were reported to reduce2-methyl-1-pyrroline stereoselectively (Mitsukara et al., 2010, Org.Biomol. Chem. 8:4533-4535).

One challenge in developing a biocatalytic route for this type ofreaction is the identification of an enzyme class that could beengineered to provide to carry out such reactions efficiently underindustrially applicable conditions. Opine dehydrogenases are a class ofoxidoreductase that act on CH—NH bonds using NADH or NADPH as co-factor.A native reaction of the opine dehydrogenases is the reductive aminationof α-keto acids with amino acids. At least five naturally occurringgenes having some homology have been identified that encode enzymeshaving the characteristic activity of opine dehydrogenase class. Thesefive enzymes include: opine dehydrogenase from Arthrobactor Sp. Strain1C (CENDH); octopine dehydrogenase from Pecten maximus (great scallop)(OpDH); ornithine synthase from Lactococcus lactis K1 (CEOS); β-alanineopine dehydrogenase from Cellana grata (BADH); and tauropinedehydrogenase from Suberites domuncula (TauDH). The crystal structure ofthe opine dehydrogenase CENDH has been determined (see Britton et al.,“Crystal structure and active site location ofN-(1-D-carboxyethyl)-L-norvaline dehydrogenase,” Nat. Struct. Biol.5(7): 593-601 (1998)). Another enzyme, N-methyl L-amino aciddehydrogenase from Pseudomonas putida (NMDH) is known to have activitysimilar to opine dehydrogenases, reacting with α-keto acids and alkylamines, but appears to have little or no sequence homology to opinedehydrogenases and amino acid dehydrogenases. NMDH has beencharacterized as belonging to a new superfamily of NAD(P) dependentoxidoreductase (see e.g., U.S. Pat. No. 7,452,704 B2; Esaki et al., FEBSJournal 2005, 272, 1117-1123).

There is a need in the art for biocatalysts and processes for usingthem, under industrially applicable conditions, for the synthesis ofchiral secondary and tertiary amines.

5. SUMMARY

The present disclosure provides novel biocatalysts and associatedmethods to use them for the synthesis of chiral secondary and tertiaryamines by direct reductive amination using an unactivated ketone and anunactivated amine as substrates. The biocatalysts of the disclosure areengineered polypeptide variants derived from a wild-type gene fromArthrobacter Sp. Strain 1C which encodes an opine dehydrogenase havingthe amino acid sequence of SEQ ID NO: 2. These engineered polypeptidesare capable of catalyzing the conversion of a ketone (includingunactivated ketone substrates such as cyclohexanone and 2-pentanone) oraldehyde substrate, and a primary or secondary amine substrate(including unactivated amine substrates such as butylamine, aniline,methylamine, and dimethylamine) to form a secondary or tertiary amineproduct compound. The enzymatic activity of these engineeredpolypeptides derived from opine dehydrogenases is referred and theengineered enzymes disclosed herein are also referred as “iminereductases” or “IREDs.” The general imine reductase activity of theIREDs is illustrated below in Scheme 1.

The engineered polypeptides having imine reductase activity of thepresent disclosure can accept a wide range of substrates. Accordingly,in the biocatalytic reaction of Scheme 1, the R¹ and R² groups of thesubstrate of formula (I) are independently selected from a hydrogenatom, or optionally substituted alkyl, alkenyl, alkynyl, alkoxy,carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl,arylalkyl, heterocycloalkyl, heteroaryl, and heteroarylalkyl; and the R³and R⁴ groups of the substrate of formula (II) are independentlyselected from a hydrogen atom, and optionally substituted alkyl,alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl,alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl,heteroaryl, and heteroarylalkyl, with the proviso that both R³ and R⁴cannot be hydrogen. Optionally, either or both of the R¹ and R² groupsof the substrate of formula (I) and the R³ and R⁴ groups of thesubstrate of formula (II), can be linked to form a 3-membered to10-membered ring. Further, the biocatalytic reaction of Scheme 1 can bean intramolecular reaction wherein at least one of the R¹ and R² groupsof the compound of formula (I) is linked to at least one of the R³ andR⁴ groups of the compound of formula (II). Also, either or both of thecarbon atom and/or the nitrogen indicated by * in the product compoundof formula (III) can be chiral. As described further herein, theengineered polypeptides having imine reductase activity exhibitstereoselectivity, thus, an imine reductase reaction of Scheme 1 can beused to establish one, two, or more, chiral centers of a productcompound of formula (III) in a single biocatalytic reaction.

In some embodiments, the present disclosure provides an engineeredpolypeptide having imine reductase activity, comprising an amino acidsequence having at least 80% sequence identity to a naturally occurringopine dehydrogenase amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 102, 104, 106, 108, and 110, and furthercomprising one or more residue differences as compared to the aminosequence of selected naturally occurring opine dehydrogenase. In someembodiments of the engineered polypeptide derived from an opinedehydrogenase, the imine reductase activity is the activity of Scheme 1,optionally, a reaction as disclosed in Table 2, and optionally, thereaction of converting compound (1b) and compound (2b) to productcompound (3d).

In some embodiments, the present disclosure provides an engineeredpolypeptide having imine reductase activity, comprising an amino acidsequence having at least 80% sequence identity to SEQ ID NO: 2 and oneor more residue differences as compared to the sequence of SEQ ID NO: 2at residue positions selected from: X111, X136, X156, X197, X198, X201,X259, X280, X292, and X293. In some embodiments, the residue differencesare selected from X111M/Q/S, X136G, X156G/I/Q/S/T/V, X197I/P,X198A/E/H/P/S, X201L, X259E/H/I/L/M/S/T, X280L, X292C/G/I/P/S/T/V/Y, andX293H/I/K/L/N/Q/T/V. In some embodiments, the engineered polypeptidecomprises a residue difference as compared to the sequence of SEQ ID NO:2 at residue position X198, wherein optionally the residue difference atposition X198 is selected from X198A, X198E, X198H, X198P, and X198S. Insome embodiments, the engineered polypeptide comprises an amino acidsequence having a residue difference at position X198 that is selectedfrom X198E, and X198H. In some embodiments, the amino acid sequence ofthe engineered polypeptides comprises at least a combination of residuedifferences selected from: (a) X111M, X156T, X198H, X259M, X280L, X292V,and X293H; (b) X156T, X197P, X198H, X259H, X280L, X292P, and X293H; (c)X111M, X136G, X156S, X197I, X198H, X201L, X259H, X280L, X292V, andX293H; (d) X197I, X198E, X259M, and X280L; (e) X156T, X197I, X198E,X201L, X259H, X280L, X292V, and X293H; (f) X111M, X136G, X198H, X259M,X280L, X292S, and X293H; and (g) X156V, X197P, X198E, X201L, X259M,X280L, and X292T.

In some embodiments, the present disclosure provides an engineeredpolypeptide having imine reductase activity, comprising an amino acidsequence having at least 80% sequence identity to SEQ ID NO: 2 and oneor more residue differences as compared to the sequence of SEQ ID NO: 2at residue positions selected from X111, X136, X156, X197, X198, X201,X259, X280, X292, and X293 (as described above), and further comprisingone or more residue differences as compared to the sequence of SEQ IDNO: 2 at residue positions selected from X4, X5, X14, X20, X29, X37,X67, X71, X74, X82, X94, X97, X100, X111, X124, X137, X141, X143, X149,X153, X154, X157, X158, X160, X163, X177, X178, X183, X184, X185, X186,X220, X223, X226, X232, X243, X246, X256, X258, X259, X260, X261, X265,X266, X270, X273, X274, X277, X279, X283, X284, X287, X288, X294, X295,X296, X297, X308, X311, X323, X324, X326, X328, X332, X353, and X356. Insome embodiments, these further residue differences are selected fromX4H/L/R, X5T, X14P, X20T, X29R/T, X37H, X67A/D, X71C/V, X74R, X82P,X94K/R/T, X97P, X100W, X111R, X124L/N, X137N, X141W, X143W, X149L,X153V/Y, X154F/M/Q/Y, X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H,X178E, X183C, X184K/Q/R, X185V, X186K/R, X220D/H, X223T, X226L, X232A/R,X243G, X246W, X256V, X258D, X259V/W, X260G, X261A/G/I/K/R/S/T,X265G/L/Y, X266T, X270G, X273W, X274M, X277A/I, X279F/L/V/Y, X283V,X284K/L/M/Y, X287S/T, X288G/S, X294A/I/V, X295R/S, X296L/N/V/W, X297A,X308F, X311C/T/V, X323C/I/M/T/V, X324L/T, X326V, X328A/G/E, X332V,X353E, X356R.

In some embodiments, the present disclosure provides an engineeredpolypeptide having imine reductase activity, comprising an amino acidsequence having at least 80% sequence identity to SEQ ID NO: 2 and oneor more residue differences as compared to the sequence of SEQ ID NO: 2at residue positions selected from X111, X136, X156, X197, X198, X201,X259, X280, X292, and X293 (as described above), and further comprisingat least a combination of residue differences selected from: (a) X29R,X184R, X223T, X261S, X284M, and X287T; (b) X29R, X157R, X184Q, X220H,X223T, X232A, X261I, X284M, X287T, X288S, X324L, X332V, and X353E; (c)X29R, X157R, X184Q, X220H, X223T, X232A, X259V, X261I, X284M, X287T,X288S, X324L, X332V, and X353E; (d) X29R, X94K, X111R, X137N, X157R,X184Q, X220H, X223T, X232A, X259V, X261I, X279V, X284M, X287T, X288S,X324L, X332V, and X353E; and (e) X29R, X94K, X111R, X137N, X157R, X184Q,X220H, X223T, X232A, X259V, X261I, X266T, X279V, X284M, X287T, X288S,X295S, X311V, X324L, X328E, X332V, and X353E.

In some embodiments, the present disclosure provides an engineeredpolypeptide having imine reductase activity, comprising an amino acidsequence having at least 80% sequence identity to SEQ ID NO: 2 and thecombination of residue differences X156T, X197I, X198E, X201L, X259H,X280L, X292V, and X293H, and further comprising one or more residuedifferences selected from X29R/T, X94K/R/T, X111R, X137N,X157D/H/L/M/N/R, X184K/Q/R, X220D/H, X223T, X232A/R, X259V/W,X261A/G/I/K/R/S/T, X266T, X279F/L/V/Y, X284K/L/M/Y, X287S/T, X288G/S,X295S, X311V, X324L/T, X328E, X332V, and X353E. In some embodiment, thesequence comprises the combination of residue differences X156T, X197I,X198E, X201L, X259H, X280L, X292V, and X293H, and further comprises atleast a combination of residue differences selected from: (a) X29R,X184R, X223T, X261S, X284M, and X287T; (b) X29R, X157R, X184Q, X220H,X223T, X232A, X261I, X284M, X287T, X288S, X324L, X332V, and X353E; (c)X29R, X157R, X184Q, X220H, X223T, X232A, X259V, X261I, X284M, X287T,X288S, X324L, X332V, and X353E; (d) X29R, X94K, X111R, X137N, X157R,X184Q, X220H, X223T, X232A, X259V, X261I, X279V, X284M, X287T, X288S,X324L, X332V, and X353E; and (e) X29R, X94K, X111R, X137N, X157R, X184Q,X220H, X223T, X232A, X259V, X261I, X266T, X279V, X284M, X287T, X288S,X295S, X311V, X324L, X328E, X332V, and X353E.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence having 70%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or greater identity to a sequence of even-numberedsequence identifiers SEQ ID NO: 4-100 and 112-750.

In another aspect, the present disclosure provides polynucleotidesencoding any of the engineered polypeptides having imine reductaseactivity disclosed herein. Exemplary polynucleotide sequences areprovided in the Sequence Listing incorporated by reference herein andinclude the sequences of odd-numbered sequence identifiers SEQ ID NO:3-99 and 111-749.

In another aspect, the polynucleotides encoding the engineeredpolypeptides having imine reductase activity of the disclosure can beincorporated into expression vectors and host cells for expression ofthe polynucleotides and the corresponding encoded polypeptides. As such,in some embodiments, the present disclosure provides methods ofpreparing the engineered polypeptides having imine reductase activity byculturing a host cell comprising the polynucleotide or expression vectorcapable of expressing an engineered polypeptide of the disclosure underconditions suitable for expression of the polypeptide. In someembodiments, the method of preparing the imine reductase polypeptide cancomprise the additional step of isolating the expressed polypeptide.

In some embodiments, the present disclosure also provides methods ofmanufacturing an engineered polypeptide having imine reductase activity,where the method can comprise: (a) synthesizing a polynucleotideencoding a polypeptide comprising an amino acid sequence selected fromthe even-numbered sequence identifiers of SEQ ID NO: 4-100 and 112-750,and having one or more residue differences as compared to SEQ ID NO:2 atresidue positions selected from: X4, X5, X14, X20, X29, X37, X67, X71,X74, X82, X94, X97, X100, X111, X124, X136, X137, X141, X143, X149,X153, X154, X156, X157, X158, X160, X163, X177, X178, X183, X184, X185,X186, X197, X198, X201, X220, X223, X226, X232, X243, X246, X256, X258,X259, X260, X261, X265, X266, X270, X273, X274, X277, X279, X280, X283,X284, X287, X288, X292, X293, X294, X295, X296, X297, X308, X311, X323,X324, X326, X328, X332, X353, and X356, and (b) expressing theengineered polypeptide encoded by the polynucleotide. As noted above,the residue differences at these positions can be selected from X4H/L/R;X5T; X14P; X20T; X29R/T; X37H; X67A/D; X71C/V; X74R; X82P; X94K/R/T;X97P; X100W; X111M/Q/R/S; X124L/N; X136G; X137N; X141W; X143W; X149L;X153V/Y; X154F/M/Q/Y; X156G/I/Q/S/T/V; X157D/H/L/M/N/R; X158K; X160N;X163T; X177C/H; X178E; X183C; X184K/Q/R; X185V; X186K/R; X197I/P;X198A/E/H/P/S; X201L; X220D/H; X223T; X226L; X232A/R; X243G; X246W;X256V; X258D; X259E/H/I/L/M/S/T/V/W; X260G; X261A/G/I/K/R/S/T;X265G/L/Y; X266T; X270G; X273W; X274M; X277A/I; X279F/L/V/Y; X280L;X283V; X284K/L/M/Y; X287S/T; X288G/S; X292C/G/I/P/S/T/V/Y;X293H/I/K/L/N/Q/T/V; X294A/I/V; X295R/S; X296L/N/V/W; X297A; X308F;X311C/T/V; X323C/I/M/T/V; X324L/T; X326V; X328A/G/E; X332V; X353E; andX356R. As further provided in the detailed description, additionalvariations can be incorporated during the synthesis of thepolynucleotide to prepare engineered imine reductase polypeptides withcorresponding differences in the expressed amino acid sequences.

In some embodiments, the engineered polypeptides having imine reductaseactivity of the present disclosure can be used in a biocatalytic processfor preparing a secondary or tertiary amine product compound of formula(III),

wherein, R¹ and R² groups are independently selected from optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl; and optionally R¹ andR² are linked to form a 3-membered to 10-membered ring; R³ and R⁴ groupsare independently selected from a hydrogen atom, and optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl, with the proviso thatboth R³ and R⁴ cannot be hydrogen; and optionally R³ and R⁴ are linkedto form a 3-membered to 10-membered ring; and optionally, the carbonatom and/or the nitrogen indicated by * is chiral. The process comprisescontacting a compound of formula (I),

wherein R¹, and R² are as defined above; and a compound of formula (II),

wherein R³, and R⁴ are as defined above; with an engineered polypeptidehaving imine reductase activity in presence of a cofactor under suitablereaction conditions.

In some embodiments of the above biocatalytic process, the engineeredpolypeptide having imine reductase activity is derived from a naturallyoccurring enzyme selected from: opine dehydrogenase from Arthrobactersp. strain 1C (SEQ ID NO: 2), D-octopine dehydrogenase from Pectenmaximus (SEQ ID NO: 102), ornithine dehydrogenase from Lactococcuslactis K1 (SEQ ID NO: 104), N-methyl-L-amino acid dehydrogenase fromPseudomonas putida (SEQ ID NO: 106), β-alanopine dehydrogenase fromCellana grata (SEQ ID NO: 108), and tauropine dehydrogenase fromSuberites domuncula (SEQ ID NO: 110). In some embodiments, theengineered polypeptide derived from the opine dehydrogenase fromArthrobacter sp. strain 1C of SEQ ID NO: 2. Any of the engineered iminereductases described herein (and exemplified by the engineered iminereductase polypeptides of even numbered sequence identifiers SEQ ID NO:4-100 and 112-750) can be used in the biocatalytic processes forpreparing a secondary or tertiary amine compound of formula (III).

In some embodiments of the process for preparing a product compound offormula (III) using an engineered imine reductase, the process furthercomprises a cofactor regeneration system capable of converting NADP⁺ toNADPH, or NAD⁺ to NADH. In some embodiments, the cofactor recyclingsystem comprises formate and formate dehydrogenase (FDH), glucose andglucose dehydrogenase (GDH), glucose-6-phosphate and glucose-6-phosphatedehydrogenase, a secondary alcohol and alcohol dehydrogenase, orphosphite and phosphite dehydrogenase. In some embodiments, the processcan be carried out, wherein the engineered imine reductase isimmobilized on a solid support.

6. DETAILED DESCRIPTION

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly indicates otherwise. Thus, for example, reference to “apolypeptide” includes more than one polypeptide. Similarly, “comprise,”“comprises,” “comprising” “include,” “includes,” and “including” areinterchangeable and not intended to be limiting. It is to be understoodthat where descriptions of various embodiments use the term“comprising,” those skilled in the art would understand that in somespecific instances, an embodiment can be alternatively described usinglanguage “consisting essentially of” or “consisting of.” It is to befurther understood that where descriptions of various embodiments usethe term “optional” or “optionally” the subsequently described event orcircumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not. It is to be understood that both the foregoing generaldescription, and the following detailed description are exemplary andexplanatory only and are not restrictive of this disclosure. The sectionheadings used herein are for organizational purposes only and not to beconstrued as limiting the subject matter described.

6.1 Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Amino Acid Three-Letter Abbreviation One-Letter Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine HIS H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

6.2 Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings:

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Polynucleotide” or “nucleic acid” refers to two or more nucleosidesthat are covalently linked together. The polynucleotide may be whollycomprised ribonucleosides (i.e., an RNA), wholly comprised of 2′deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), itmay include one or more modified and/or synthetic nucleobases, such as,for example, inosine, xanthine, hypoxanthine, etc. Preferably, suchmodified or synthetic nucleobases will be encoding nucleobases.

“Opine dehydrogenase activity,” as used herein, refers to an enzymaticactivity in which a carbonyl group of a 2-ketoacid (e.g., pyruvate) andan amino group of a neutral L-amino acid (e.g., L-norvaline) areconverted to a secondary amine dicarboxylate compound (e.g., such asN-[1-(R)-(carboxy)ethyl]-(S)-norvaline).

“Opine dehydrogenase,” as used herein refers to an enzyme having opinedehydrogenase activity. Opine dehydrogenase includes but is not limitedto the following naturally occurring enzymes: opine dehydrogenase fromArthrobacter Sp. Strain 1C (CENDH) (SEQ ID NO: 2); octopinedehydrogenase from Pecten maximus (OpDH) (SEQ ID NO: 102); ornithinesynthase from Lactococcus lactis K1 (CEOS) (SEQ ID NO: 104); N-methylL-amino acid dehydrogenase from Pseudomonas putida (NMDH) (SEQ ID NO:106); β-alanopine dehydrogenase from Cellana grata (BADH) (SEQ ID NO:108); tauropine dehydrogenase from Suberites domuncula (TauDH) (SEQ IDNO: 110); saccharopine dehydrogenase from Yarrowia lipolytica (SacDH)(UniProtKB entry: P38997, entry name: LYS1_YARLI); and D-nopalinedehydrogenase from Agrobacterium tumefaciens (strain T37) (UniProtKBentry: P00386, entry name: DHNO_AGRT7).

“Imine reductase activity,” as used herein, refers to an enzymaticactivity in which a carbonyl group of a ketone or aldehyde and an aminogroup a primary or secondary amine (wherein the carbonyl and aminogroups can be on separate compounds or the same compound) are convertedto a secondary or tertiary amine product compound, in the presence ofco-factor NAD(P)H, as illustrated in Scheme 1.

“Imine reductase” or “IRED,” as used herein, refers to an enzyme havingimine reductase activity. It is to be understood that imine reductasesare not limited to engineered polypeptides derived from the wild-typeopine dehydrogenase from Arthrobacter Sp. Strain 1C, but may includeother enzymes having imine reductase activity, including engineeredpolypeptides derived from other opine dehydrogenase enzymes, such asoctopine dehydrogenase from Pecten maximus (OpDH), ornithine synthasefrom Lactococcus lactis K1 (CEOS), β-alanopine dehydrogenase fromCellana grata (BADH), tauropine dehydrogenase from Suberites domuncula(TauDH); and N-methyl L-amino acid dehydrogenase from Pseudomonas putida(NMDH); or an engineered enzyme derived from a wild-type enzyme havingimine reductase activity. Imine reductases as used herein includenaturally occurring (wild-type) imine reductase as well as non-naturallyoccurring engineered polypeptides generated by human manipulation.

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” or “non-naturally occurring” when usedwith reference to, e.g., a cell, nucleic acid, or polypeptide, refers toa material, or a material corresponding to the natural or native form ofthe material, that has been modified in a manner that would nototherwise exist in nature, or is identical thereto but produced orderived from synthetic materials and/or by manipulation usingrecombinant techniques. Non-limiting examples include, among others,recombinant cells expressing genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence for optimal alignment of the two sequences. Thepercentage may be calculated by determining the number of positions atwhich the identical nucleic acid base or amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Alternatively, the percentage may becalculated by determining the number of positions at which either theidentical nucleic acid base or amino acid residue occurs in bothsequences or a nucleic acid base or amino acid residue is aligned with agap to yield the number of matched positions, dividing the number ofmatched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Those of skill in the art appreciate that there aremany established algorithms available to align two sequences. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch,1970, J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1995Supplement) (Ausubel)). Examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplarydetermination of sequence alignment and % sequence identity can employthe BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence. For instance, a “reference sequence based on SEQID NO:4 having at the residue corresponding to X14 a valine” or X14Vrefers to a reference sequence in which the corresponding residue at X14in SEQ ID NO:4, which is a tyrosine, has been changed to valine.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredimine reductase, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned.

“Amino acid difference” or “residue difference” refers to a change inthe amino acid residue at a position of a polypeptide sequence relativeto the amino acid residue at a corresponding position in a referencesequence. The positions of amino acid differences generally are referredto herein as “Xn,” where n refers to the corresponding position in thereference sequence upon which the residue difference is based. Forexample, a “residue difference at position X25 as compared to SEQ ID NO:2” refers to a change of the amino acid residue at the polypeptideposition corresponding to position 25 of SEQ ID NO:2. Thus, if thereference polypeptide of SEQ ID NO: 2 has a valine at position 25, thena “residue difference at position X25 as compared to SEQ ID NO:2” anamino acid substitution of any residue other than valine at the positionof the polypeptide corresponding to position 25 of SEQ ID NO: 2. In mostinstances herein, the specific amino acid residue difference at aposition is indicated as “XnY” where “Xn” specified the correspondingposition as described above, and “Y” is the single letter identifier ofthe amino acid found in the engineered polypeptide (i.e., the differentresidue than in the reference polypeptide). In some embodiments, theremore than one amino acid can appear in a specified residue position, thealternative amino acids can be listed in the form XnY/Z, where Y and Zrepresent alternate amino acid residues. In some instances (e.g., inTables 3A, 3B, 3C, 3D and 3E), the present disclosure also providesspecific amino acid differences denoted by the conventional notation“AnB”, where A is the single letter identifier of the residue in thereference sequence, “n” is the number of the residue position in thereference sequence, and B is the single letter identifier of the residuesubstitution in the sequence of the engineered polypeptide. Furthermore,in some instances, a polypeptide of the present disclosure can includeone or more amino acid residue differences relative to a referencesequence, which is indicated by a list of the specified positions wherechanges are made relative to the reference sequence. The presentdisclosure includes engineered polypeptide sequences comprising one ormore amino acid differences that include either/or both conservative andnon-conservative amino acid substitutions.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid, e.g., alanine, valine, leucine, and isoleucine; an amino acid withhydroxyl side chain is substituted with another amino acid with ahydroxyl side chain, e.g., serine and threonine; an amino acid havingaromatic side chains is substituted with another amino acid having anaromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, andhistidine; an amino acid with a basic side chain is substituted withanother amino acid with a basic side chain, e.g., lysine and arginine;an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain, e.g., aspartic acid or glutamicacid; and a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively. Exemplaryconservative substitutions are provided in Table 1 below.

TABLE 1 Residue Possible Conservative Substitutions A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) N, Q, S, T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C,P None

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine), (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered imine reductase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered imine reductase enzymes comprise insertions ofone or more amino acids to the naturally occurring polypeptide havingimine reductase activity as well as insertions of one or more aminoacids to other improved imine reductase polypeptides. Insertions can bein the internal portions of the polypeptide, or to the carboxy or aminoterminus. Insertions as used herein include fusion proteins as is knownin the art. The insertion can be a contiguous segment of amino acids orseparated by one or more of the amino acids in the naturally occurringpolypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length imine reductasepolypeptide, for example the polypeptide of SEQ ID NO:2 or engineeredimine reductase of SEQ ID NO:96.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The engineered imine reductase enzymes may be present withina cell, present in the cellular medium, or prepared in various forms,such as lysates or isolated preparations. As such, in some embodiments,the engineered imine reductase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure imine reductase compositionwill comprise about 60% or more, about 70% or more, about 80% or more,about 90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedengineered imine reductase polypeptide is a substantially purepolypeptide composition.

“Stereoselective” refers to a preference for formation of onestereoisomer over another in a chemical or enzymatic reaction.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a chemical or enzymatic reaction thatis capable of converting a substrate or substrates, e.g., substratecompounds (1e) and (2b), to the corresponding amine product, e.g.,compound (3i), with at least about 85% stereomeric excess.

“Improved enzyme property” refers to an imine reductase polypeptide thatexhibits an improvement in any enzyme property as compared to areference imine reductase. For the engineered imine reductasepolypeptides described herein, the comparison is generally made to thewild-type enzyme from which the imine reductase is derived, although insome embodiments, the reference enzyme can be another improvedengineered imine reductase. Enzyme properties for which improvement isdesirable include, but are not limited to, enzymatic activity (which canbe expressed in terms of percent conversion of the substrate), thermostability, solvent stability, pH activity profile, cofactorrequirements, refractoriness to inhibitors (e.g., substrate or productinhibition), stereospecificity, and stereoselectivity (includingenantioselectivity).

“Increased enzymatic activity” refers to an improved property of theengineered imine reductase polypeptides, which can be represented by anincrease in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount of iminereductase) as compared to the reference imine reductase enzyme.Exemplary methods to determine enzyme activity are provided in theExamples. Any property relating to enzyme activity may be affected,including the classical enzyme properties of K_(m), V_(max) or k_(cat),changes of which can lead to increased enzymatic activity. Improvementsin enzyme activity can be from about 1.2 times the enzymatic activity ofthe corresponding wild-type enzyme, to as much as 2 times, 5 times, 10times, 20 times, 25 times, 50 times or more enzymatic activity than thenaturally occurring or another engineered imine reductase from which theimine reductase polypeptides were derived. Imine reductase activity canbe measured by any one of standard assays, such as by monitoring changesin properties of substrates, cofactors, or products. In someembodiments, the amount of products generated can be measured by LiquidChromatography-Mass Spectrometry (LC-MS). Comparisons of enzymeactivities are made using a defined preparation of enzyme, a definedassay under a set condition, and one or more defined substrates, asfurther described in detail herein. Generally, when lysates arecompared, the numbers of cells and the amount of protein assayed aredetermined as well as use of identical expression systems and identicalhost cells to minimize variations in amount of enzyme produced by thehost cells and present in the lysates.

“Conversion” refers to the enzymatic conversion of the substrate(s) tothe corresponding product(s). “Percent conversion” refers to the percentof the substrate that is converted to the product within a period oftime under specified conditions. Thus, the “enzymatic activity” or“activity” of a imine reductase polypeptide can be expressed as “percentconversion” of the substrate to the product.

“Thermostable” refers to a imine reductase polypeptide that maintainssimilar activity (more than 60% to 80% for example) after exposure toelevated temperatures (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the wild-type enzyme.

“Solvent stable” refers to an imine reductase polypeptide that maintainssimilar activity (more than e.g., 60% to 80%) after exposure to varyingconcentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol,dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran,acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for aperiod of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme.

“Thermo- and solvent stable” refers to an imine reductase polypeptidethat is both thermostable and solvent stable.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad.Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychliket al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, NucleicAcids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered imine reductase enzyme of the present disclosure.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding theimine reductase enzymes may be codon optimized for optimal productionfrom the host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the nucleic acid sequence encoding thepolypeptide. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Suitable reaction conditions” refer to those conditions in thebiocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, temperature, pH, buffers,co-solvents, etc.) under which an imine reductase polypeptide of thepresent disclosure is capable of converting a substrate compound to aproduct compound (e.g., conversion of compound (2) to compound (1)).Exemplary “suitable reaction conditions” are provided in the presentdisclosure and illustrated by the Examples.

“Cofactor regeneration system” or “cofactor recycling system” refers toa set of reactants that participate in a reaction that reduces theoxidized form of the cofactor (e.g., NADP⁺ to NADPH). Cofactors oxidizedby the imine reductase catalyzed reductive amination of the ketonesubstrate are regenerated in reduced form by the cofactor regenerationsystem. Cofactor regeneration systems comprise a stoichiometricreductant that is a source of reducing hydrogen equivalents and iscapable of reducing the oxidized form of the cofactor. The cofactorregeneration system may further comprise a catalyst, for example anenzyme catalyst that catalyzes the reduction of the oxidized form of thecofactor by the reductant. Cofactor regeneration systems to regenerateNADH or NADPH from NAD⁺ or NADP⁺, respectively, are known in the art andmay be used in the methods described herein.

“Formate dehydrogenase” and “FDH” are used interchangeably herein torefer to an NAD⁺ or NADP⁺-dependent enzyme that catalyzes the conversionof formate and NAD⁺ or NADP⁺ to carbon dioxide and NADH or NADPH,respectively.

“Loading”, such as in “compound loading” or “enzyme loading” or“cofactor loading” refers to the concentration or amount of a componentin a reaction mixture at the start of the reaction.

“Substrate” in the context of a biocatalyst mediated process refers tothe compound or molecule acted on by the biocatalyst. For example, animine reductase biocatalyst used in the reductive amination processesdisclosed herein there is a ketone (or aldehyde) substrate of formula(I), such as cyclohexanone, and an amine substrate of formula (II), suchas butylamine.

“Product” in the context of a biocatalyst mediated process refers to thecompound or molecule resulting from the action of the biocatalyst. Forexample, an exemplary product for an imine reductase biocatalyst used ina process disclosed herein is a secondary or tertiary amine compound,such as a compound of formula (III).

“Alkyl” refers to saturated hydrocarbon groups of from 1 to 18 carbonatoms inclusively, either straight chained or branched, more preferablyfrom 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbonatoms inclusively. An alkyl with a specified number of carbon atoms isdenoted in parenthesis, e.g., (C₁-C₆)alkyl refers to an alkyl of 1 to 6carbon atoms.

“Alkylene” refers to a straight or branched chain divalent hydrocarbonradical having from 1 to 18 carbon atoms inclusively, more preferablyfrom 1 to 8 carbon atoms inclusively, and most preferably 1 to 6 carbonatoms inclusively.

“Alkenyl” refers to groups of from 2 to 12 carbon atoms inclusively,either straight or branched containing at least one double bond butoptionally containing more than one double bond.

“Alkenylene” refers to a straight or branched chain divalent hydrocarbonradical having 2 to 12 carbon atoms inclusively and one or morecarbon-carbon double bonds, more preferably from 2 to 8 carbon atomsinclusively, and most preferably 2 to 6 carbon atoms inclusively.

“Alkynyl” refers to groups of from 2 to 12 carbon atoms inclusively,either straight or branched containing at least one triple bond butoptionally containing more than one triple bond, and additionallyoptionally containing one or more double bonded moieties.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 12 carbon atomsinclusively having a single cyclic ring or multiple condensed ringswhich can be optionally substituted with from 1 to 3 alkyl groups.Exemplary cycloalkyl groups include, but are not limited to, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl,1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and thelike, or multiple ring structures, including bridged ring systems, suchas adamantyl, and the like.

“Cycloalkylalkyl” refers to an alkyl substituted with a cycloalkyl,i.e., cycloalkyl-alkyl- groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 3 to 12 carbon atomsinclusively in the cycloalkyl moiety. Such cycloalkylalkyl groups areexemplified by cyclopropylmethyl, cyclohexylethyl and the like.

“Aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to12 carbon atoms inclusively having a single ring (e.g., phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl). Exemplary arylsinclude phenyl, pyridyl, naphthyl and the like.

“Arylalkyl” refers to an alkyl substituted with an aryl, i.e.,aryl-alkyl- groups, preferably having from 1 to 6 carbon atomsinclusively in the alkyl moiety and from 6 to 12 carbon atomsinclusively in the aryl moiety. Such arylalkyl groups are exemplified bybenzyl, phenethyl and the like.

“Heteroalkyl, “heteroalkenyl,” and heteroalkynyl,” refer to alkyl,alkenyl and alkynyl as defined herein in which one or more of the carbonatoms are each independently replaced with the same or differentheteroatoms or heteroatomic groups. Heteroatoms and/or heteroatomicgroups which can replace the carbon atoms include, but are not limitedto, —O—, —S—, —S—O—, —NR^(γ)—, —PH—, —S(O)—, —S(O)₂—, —S(O)NR^(γ)—,—S(O)₂NR^(γ)—, and the like, including combinations thereof, where eachR¹ is independently selected from hydrogen, alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

“Heteroaryl” refers to an aromatic heterocyclic group of from 1 to 10carbon atoms inclusively and 1 to 4 heteroatoms inclusively selectedfrom oxygen, nitrogen and sulfur within the ring. Such heteroaryl groupscan have a single ring (e.g., pyridyl or furyl) or multiple condensedrings (e.g., indolizinyl or benzothienyl).

“Heteroarylalkyl” refers to an alkyl substituted with a heteroaryl,i.e., heteroaryl-alkyl- groups, preferably having from 1 to 6 carbonatoms inclusively in the alkyl moiety and from 5 to 12 ring atomsinclusively in the heteroaryl moiety. Such heteroarylalkyl groups areexemplified by pyridylmethyl and the like.

“Heterocycle”, “heterocyclic” and interchangeably “heterocycloalkyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, from 2 to 10 carbon ring atoms inclusively andfrom 1 to 4 hetero ring atoms inclusively selected from nitrogen, sulfuror oxygen within the ring. Such heterocyclic groups can have a singlering (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings(e.g., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples ofheterocycles include, but are not limited to, furan, thiophene,thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, isothiazole, phenazine,isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline,piperidine, piperazine, pyrrolidine, indoline and the like.

“Heterocycloalkylalkyl” refers to an alkyl substituted with aheterocycloalkyl, i.e., heterocycloalkyl-alkyl- groups, preferablyhaving from 1 to 6 carbon atoms inclusively in the alkyl moiety and from3 to 12 ring atoms inclusively in the heterocycloalkyl moiety.

“Oxy” refers to a divalent group —O—, which may have varioussubstituents to form different oxy groups, including ethers and esters.

“Alkoxy” or “alkyloxy” are used interchangeably herein to refer to thegroup —OR^(ζ), wherein W is an alkyl group, including optionallysubstituted alkyl groups.

“Aryloxy” as used herein refer to the group —OR wherein R is an arylgroup as defined above including optionally substituted aryl groups asalso defined herein.

“Carboxy” refers to —COOH.

“Carboxyalkyl” refers to an alkyl substituted with a carboxy group.

“Carbonyl” refers to the group —C(O)—. Substituted carbonyl refers tothe group R^(η)—C(O)—R^(η), where each R^(η) is independently selectedfrom optionally substituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy,carboxy, aryl, aryloxy, heteroaryl, heteroarylalkyl, acyl,alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Typicalsubstituted carbonyl groups including acids, ketones, aldehydes, amides,esters, acyl halides, thioesters, and the like.

“Amino” refers to the group —NH₂. Substituted amino refers to the group—NHR^(η), NR^(η)R^(η), and NR^(η)R^(η)R^(η), where each R^(η) isindependently selected from optionally substituted alkyl, cycloalkyl,cycloheteroalkyl, alkoxy, carboxy, aryl, aryloxy, heteroaryl,heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, andthe like. Typical amino groups include, but are limited to,dimethylamino, diethylamino, trimethylammonium, triethylammonium,methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

“Aminoalkyl” refers to an alkyl group in which one or more of thehydrogen atoms are replaced with an amino group, including a substitutedamino group.

“Aminocarbonyl” refers to a carbonyl group substituted with an aminogroup, including a substituted amino group, as defined herein, andincludes amides.

“Aminocarbonylalkyl” refers to an alkyl substituted with anaminocarbonyl group, as defined herein.

“Halogen” or “halo” refers to fluoro, chloro, bromo and iodo.

“Haloalkyl” refers to an alkyl group in which one or more of thehydrogen atoms are replaced with a halogen. Thus, the term “haloalkyl”is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. upto perhaloalkyls. For example, the expression “(C₁ C₂) haloalkyl”includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl,1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl,perfluoroethyl, etc.

“Hydroxy” refers to —OH.

“Hydroxyalkyl” refers to an alkyl substituted with one or more hydroxygroup.

“Thio” or “sulfanyl” refers to —SH. Substituted thio or sulfanyl refersto —S—R^(η), where R^(η) is an alkyl, aryl or other suitablesubstituent.

“Alkylthio” refers to —SR^(ζ), where R^(ζ) is an alkyl, which can beoptionally substituted. Typical alkylthio group include, but are notlimited to, methylthio, ethylthio, n-propylthio, and the like.

“Alkylthioalkyl” refers to an alkyl substituted with an alkylthio group,—SR^(ζ), where R^(ζ) is an alkyl, which can be optionally substituted.

“Sulfonyl” refers to —SO₂—. Substituted sulfonyl refers to —SO₂—R^(η),where R^(η) is an alkyl, aryl or other suitable substituent.

“Alkylsulfonyl” refers to —SO₂—R^(ζ), where R^(ζ) is an alkyl, which canbe optionally substituted. Typical alkylsulfonyl groups include, but arenot limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and thelike.

“Alkylsulfonylalkyl” refers to an alkyl substituted with analkylsulfonyl group, —SO₂—R^(ζ), where R^(ζ) is an alkyl, which can beoptionally substituted.

“Membered ring” is meant to embrace any cyclic structure. The numberpreceding the term “membered” denotes the number of skeletal atoms thatconstitute the ring. Thus, for example, cyclohexyl, pyridine, pyran andthiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, andthiophene are 5-membered rings.

“Fused bicyclic ring” refers to both unsubstituted and substitutedcarbocyclic and/or heterocyclic ring moieties having 5 or 8 atoms ineach ring, the rings having 2 common atoms.

“Optionally substituted” as used herein with respect to the foregoingchemical groups means that positions of the chemical group occupied byhydrogen can be substituted with another atom, such as carbon, oxygen,nitrogen, or sulfur, or a chemical group, exemplified by, but notlimited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substitutedalkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo,methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substitutedalkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio,alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substitutedcarboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino,sulfonamido, substituted sulfonamido, cyano, amino, substituted amino,alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo,hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl,arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substitutedheteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl,cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle,(heterocycle)oxy, and (heterocycle)alkyl; where preferred heteroatomsare oxygen, nitrogen, and sulfur. Additionally, where open valencesexist on these substitute chemical groups they can be furthersubstituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocyclegroups, that where these open valences exist on carbon they can befurther substituted by halogen and by oxygen-, nitrogen-, orsulfur-bonded substituents, and where multiple such open valences exist,these groups can be joined to form a ring, either by direct formation ofa bond or by formation of bonds to a new heteroatom, preferably oxygen,nitrogen, or sulfur. It is further contemplated that the abovesubstitutions can be made provided that replacing the hydrogen with thesubstituent does not introduce unacceptable instability to the moleculesof the present disclosure, and is otherwise chemically reasonable. Oneof ordinary skill in the art would understand that with respect to anychemical group described as optionally substituted, only stericallypractical and/or synthetically feasible chemical groups are meant to beincluded. Finally, “optionally substituted” as used herein refers to allsubsequent modifiers in a term or series of chemical groups. Forexample, in the term “optionally substituted arylalkyl,” the “alkyl”portion and the “aryl” portion of the molecule may or may not besubstituted, and for the series “optionally substituted alkyl,cycloalkyl, aryl and heteroaryl,” the alkyl, cycloalkyl, aryl, andheteroaryl groups, independently of the others, may or may not besubstituted.

6.3 Engineered Imine Reductase (IRED) Polypeptides

The present disclosure provides engineered polypeptides having iminereductase activity, polynucleotides encoding the polypeptides; methodsof preparing the polypeptides, and methods for using the polypeptides.Where the description relates to polypeptides, it is to be understoodthat it also describes the polynucleotides encoding the polypeptides.

As noted above, imine reductases belong to a class of enzymes thatcatalyze the reductive amination of a ketone substrate and a primary orsecondary amine substrate to a secondary or tertiary amine product, asillustrated by Scheme 1 (see above for Scheme and group structures forcompounds of formula (I), (II), and (III)).

The opine dehydrogenase from Arthrobacter Sp. Strain 1C (also referredto herein as “CENDH”) having the amino acid sequence of SEQ ID NO: 2,naturally catalyzes the conversion of ketone substrate, pyruvate and theamino acid substrate, L-2-amino pentanoic acid (or “L-norvaline”) to theproduct (2S)-2-((1-carboxyethyl)amino)pentanoic acid. CENDH alsocatalyzes the reaction of pyruvate with the amino acid substrates,L-ornithine, and β-alanine, and structurally similar amino sulfonic acidsubstrate, taurine. In addition, CENDH was found to catalyze theconversion of the unactivated ketone substrate, cyclohexanone (ratherthan pyruvate) and its natural amine substrate, L-norvaline, to thesecondary amine product, (S)-2-(cyclohexylamino)pentanoic acid. CENDHalso was found to catalyze the conversion of its natural ketonesubstrate pyruvate with the primary amines butylamine, ethylamine, andisopropylamine, to their respective 2-(alkylamino)propanoic acidsecondary amine products. CENDH, however, did not exhibit any activityfor the conversion of pyruvate with secondary amines, such asdimethylamine. Furthermore, CENDH did not show any imine reductaseactivity with the unactivated ketone substrate, cyclohexanone, when usedtogether with the unactivated primary amine substrate, butylamine.

In the present disclosure, engineered imine reductases are describedthat overcome the deficiencies of the wild-type opine dehydrogenaseCENDH. The engineered imine reductase polypeptides derived from thewild-type enzyme of Arthrobacter Sp. Strain 1C are capable ofefficiently converting pyruvate and L-norvaline to the product(2S)-2-((1-carboxyethyl)amino)pentanoic acid, but also capable ofefficiently converting a range of ketone substrate compounds of formula(I) and amine substrate compounds of formula (II), to the secondary andtertiary amine product compounds of formula (III) as shown by conversionreactions (a) through (o) which are listed below in Table 2.

TABLE 2 Conversion Reaction Substrate Compound of Substrate CompoundProduct Compound(s) of ID formula (I) of formula (II) formula (III) (a)

(b)

(c)

(d)

(e)

H₂N— (2c)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

(m)

(n)

(o)

Significantly, the present disclosure identifies amino acid residuepositions and corresponding mutations in the CENDH polypeptide of SEQ IDNO: 2 that improve its enzyme properties as compared to the naturallyoccurring enzyme, including among others, imine reductase activity,substrate specificity, and selectivity. In particular, the presentdisclosure provides engineered IRED polypeptides capable of catalyzingreductive amination reactions such as those of Table 2, i.e., thereductive amination of ketone substrate compounds of formula (I) (e.g.,cyclohexanone) with primary and secondary amine substrate compounds offormula (II) thereby producing secondary or tertiary amine compounds offormula (III).

In some embodiments, the engineered imine reductase polypeptides show anincreased activity in the conversion of the ketone substrate of formula(I) and amine substrate of formula (II) to an amine product of formula(III), in a defined time with the same amount of enzyme as compared tothe wild-type enzyme, CENDH. In some embodiments, the engineered iminereductase polypeptide has at least about 1.2 fold, 1.5 fold, 2 fold, 3fold, 4 fold, 5 fold, or 10 fold or more the activity as compared to thewild-type CENDH polypeptide represented by SEQ ID NO:2 under suitablereaction conditions.

In some embodiments, the engineered imine reductase polypeptides exhibitan imine reductase activity in the conversion of a ketone substrate offormula (I) and an amine substrate of formula (II) to an amine productof formula (III), for which the wild-type polypeptide, CENDH, has nodetectable activity.

The product compounds of formula (III) produced by the engineered iminereductase polypeptides can be a secondary or tertiary amine compoundshaving one or more chiral centers. In some embodiments, the engineeredimine reductase polypeptides are capable of converting the ketone andamine substrate compounds of formula (I) and formula (II), to a chiralamine product compound of formula (III), in an enantiomeric excess ordiastereomeric excess of greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or greater.

In some embodiments, the engineered imine reductase polypeptides arecapable of converting the ketone and amine substrate compounds offormula (I) and formula (II) with increased tolerance for the presenceof one or both of these substrate compounds relative to the tolerance ofthe reference polypeptide of SEQ ID NO: 2 under suitable reactionconditions. Thus, in some embodiments the engineered imine reductasepolypeptides are capable of converting the ketone and amine substratecompounds of formula (I) and formula (II) at a substrate loadingconcentration of at least about 10 g/L, about 20 g/L, about 30 g/L,about 40 g/L, about 50 g/L, about 70 g/L, about 100 g/L, about 125 g/L,about 150 g/L. about 175 g/L or about 200 g/L or more with a percentconversion of at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, in areaction time of about 120 h or less, 72 h or less, about 48 h or less,about 36 h or less, or about 24 h less, under suitable reactionconditions.

The suitable reaction conditions under which the above-describedimproved properties of the engineered polypeptides carry out theconversion can be determined with respect to concentrations or amountsof polypeptide, substrate, cofactor (e.g., NAD(P)H), coenzyme (e.g., FDHor GDH), buffer, co-solvent, pH, temperature, reaction time, and/orconditions with the polypeptide immobilized on a solid support, asfurther described below and in the Examples.

The present disclosure provides numerous exemplary engineeredpolypeptides having imine reductase activity. These exemplarypolypeptides were evolved from the wild-type CENDH of SEQ ID NO: 2 andexhibit improved properties, particularly increased activity andstability in the conversion of various ketone and amine substrates,including the conversion of compounds (1b) and (2b) to the amine productcompound (3d), the conversion of compounds (1i) and (2b) to the amineproduct compound (3n), and the conversion of compounds (1j) and (2b) tothe amine product compound (3o). These exemplary engineered polypeptideshaving imine reductase activity have amino acid sequences (provided inthe accompanying Sequence Listing as even-numbered sequence identifiersof SEQ ID NO: 4-100, and 112-750) that include one or more residuedifferences as compared to SEQ ID NO:2 at the following residuepositions: X4, X5, X14, X20, X29, X37, X67, X71, X74, X82, X94, X97,X100, X111, X124, X136, X137, X141, X143, X149, X153, X154, X156, X157,X158, X160, X163, X177, X178, X183, X184, X185, X186, X197, X198, X201,X220, X223, X226, X232, X243, X246, X256, X258, X259, X260, X261, X265,X266, X270, X273, X274, X277, X279, X280, X283, X284, X287, X288, X292,X293, X294, X295, X296, X297, X308, X311, X323, X324, X326, X328, X332,X353, and X356. The specific amino acid differences at each of thesepositions that are associated with the improved properties of theexemplary polypeptides of Tables 3A-3J include: X4H/L/R; X5T; X14P;X20T; X29R/T; X37H; X67A/D; X71C/V; X74R; X82P; X94K/R/T; X97P; X100W;X111M/Q/R/S; X124L/N; X136G; X137N; X141W; X143W; X149L; X153V/Y;X154F/M/Q/Y; X156G/I/Q/S/T/V; X157D/H/L/M/N/R; X158K; X160N; X163T;X177C/H; X178E; X183C; X184K/Q/R; X185V; X186K/R; X197I/P;X198A/E/H/P/S; X201L; X220D/H; X223T; X226L; X232A/R; X243G; X246W;X256V; X258D; X259E/H/I/L/M/S/T/V/W; X260G; X261A/G/I/K/R/S/T;X265G/L/Y; X266T; X270G; X273W; X274M; X277A/I; X279F/L/V/Y; X280L;X283V; X284K/L/M/Y; X287S/T; X288G/S; X292C/G/I/P/S/T/V/Y;X293H/I/K/L/N/O/T/V; X294A/I/V; X295R/S; X296L/N/V/W; X297A; X308F;X311C/T/V; X323C/I/M/T/V; X324L/T; X326V; X328A/G/E; X332V; X353E; andX356R.

The structure and function information for exemplary non-naturallyoccurring (or engineered) imine reductase polypeptides of the presentdisclosure are based on five different high-throughput (HTP) screeningassays used in the directed evolution of these enzymes: (1) theconversion of the ketone and amine substrate compounds (1a) and (2b) tothe amine product compound (3b); (2) the conversion of the ketone andamine substrate compounds (1b) and (2a) to the amine product compound(3c); (3) the conversion of the ketone and amine substrate compounds(1b) and (2b) to the amine product compound (3d); (4) the conversion ofthe ketone and amine substrate compounds (1i) and (2b) to the amineproduct compound (3n); and (5) the conversion of the ketone and aminesubstrate compounds (1j) and (2b) to the amine product compound (3o).The results of these HTP screening assays which are shown below inTables 3A, 3B, and 3F-3J. The odd numbered sequence identifiers (i.e.,SEQ ID NOs) refer to the nucleotide sequence encoding the amino acidsequence provided by the even numbered SEQ ID NOs, and the sequences areprovided in the electronic sequence listing file accompanying thisdisclosure, which is hereby incorporated by reference herein. The aminoacid residue differences listed in Tables 3A, and 3B, are based oncomparison to the reference sequence of SEQ ID NO: 2, which is naturallyoccurring amino acid sequence of the opine dehydrogenase fromArthrobacter Sp. Strain 1C, CENDH. The amino acid residue differenceslisted in Tables 3F-3J, are based on comparison to the referencesequence of SEQ ID NO: 96, which is the amino acid sequence of anengineered polypeptide having the following 7 residue differences ascompared to the opine dehydrogenase from Arthrobacter Sp. Strain 1C,CENDH: K156T, V197I, N198E, M201L, Y259H, Y280L, R292V, and Y293H.

The activity of the engineered imine reductase polypeptides relative tothe reference polypeptide of SEQ ID NO: 2 was determined using one ormore of the five high-throughput (HTP) assays as the primary screen: (1)conversion of the substrates pyruvate (compound (1a)) and butylamine(compound (2b)) to the product 2-(butylamino)propanoic acid (compound(3b)); (2) conversion of the substrates cyclohexanone (compound (1b))and L-norvaline (compound (2a)) to the product(S)-2-(cyclohexylamino)pentanoic acid (compound (3c)); (3) conversion ofthe substrates cyclohexanone and butylamine to the productN-butylcyclohexanamine (compound (3d)); (4) conversion of the substrates5-methoxy-3,4-dihydronaphthalen-2(1H)-one (compound (1i)) and butylamine(compound (2b)) to the secondary amine productN-butyl-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-amine (compound (3n));and (5) conversion of the substrates2-(3,4-dimethoxyphenethoxy)cyclohexanone (compound (1j) and butylamine(compound (2b)) to the secondary amine productN-butyl-2-(3,4-dimethoxyphenethoxy)cyclohexanamine (compound (3o)). TheHTP assay values in Tables 3A and 3B were determined using E. coli clearcell lysates in 96 well-plate format of ˜300 μL volume per wellfollowing assay reaction conditions as noted in the Tables and theExamples. The HTP assay values in Tables 3F-3J were determined using E.coli clear cell lysates in 96 well-plate format of ˜100 μL volume perwell following assay reaction conditions as noted in the Tables and theExamples.

In some instances, shake flask powder (SFP) assays also were used toassess the activity of the engineered imine reductases, the results ofwhich are provided in Tables 3C and 3D. The SFP preparations provide amore purified powder of the engineered polypeptides that are up to about30% of total protein. The SFP assay reaction conditions are noted in theTables and Examples. In addition, Tables 3C, 3D and 3E provide SFP assayresults for engineered imine reductase polypeptides when used with othercombinations of ketone and amine substrates shown in Table 2, includingthe ketone substrates, 2-methoxy-cyclohexanone, cyclopentanone, andacetophenone, hydroxyacetone, and the amine substrates, methylamine,dimethylamine, and aniline. The engineered imine reductases were alsoassayed for their selectivity for certain enantiomeric or diastereomericproducts by measuring the ratio of products formed in the reactions,such as the diastereomeric product compounds (3h) and (3i), as shown inTables 3C and 3E.

TABLE 3A Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ ID Increased Activity¹ Increased Activity¹ NO:Amino Acid Differences cyclohexanone/L-norvaline² pyruvate/butylamine³(nt/aa) (relative to SEQ ID NO: 2) (relative to SEQ ID NO: 2) (relativeto SEQ ID NO: 2) 3/4 N198A; ++ − 5/6 N198H; ++++ − 7/8 K156I; − +  9/10V197I; + − 11/12 K156V; − ++ 13/14 Y259T; − +++ 15/16 N198P; + − 17/18K156T; − ++ 19/20 K156Q; − + 21/22 K156S; − ++ 23/24 Y259S; − ++ 25/26Y259M; − ++ 27/28 K156G; − + 29/30 S136G; − + 31/32 Y259H; − +++ 33/34M201L; + − 35/36 Y280L; − + 37/38 Y259I; − + 39/40 N198S; ++ − 41/42Y259L; − + 43/44 Y259E; − + 45/46 A111S; − + 47/48 Y293T; +++ − 49/50A111Q; − +++ 51/52 Y293Q; ++ − 53/54 Y293L; ++ − 55/56 R292I; +++ −57/58 R292V; +++++ − 59/60 Y293V; +++++ − 61/62 R292T; +++++ − 63/64Y293H; ++++ − 65/66 R292G; +++ − 67/68 R292P; ++++ − 69/70 R292C; +++ −71/72 R292Y; +++ − 73/74 R292S; +++++ − 75/76 Y293I; ++++ − 77/78A111M; + +++ 79/80 Y293K; ++ − 81/82 Y293N; ++ − 83/84 V197P; + + 85/86N198E; ++ − ¹Levels of increased activity were determined relative tothe reference polypeptide of SEQ ID NO: 2 and defined as follows: “−” =activity less than or equal to reference polypeptide; “+” = at least1.1-fold but less than 2.5-fold increased activity; “++” = at least2.5-fold but less than 5-fold increased activity; “+++” = at least5-fold increased activity but less than 10-fold; “++++” = at least 10fold but less than 15-fold; and “+++++” at least 15-fold but less than20-fold. ²cyclohexanone/L-norvaline assay conditions: E. coli cellsexpressing the polypeptide variant gene of interest were pelleted,placed in 96-well plates and lysed in 250 μL lysis buffer (1 g/Llysozyme and 1 g/L PMBS in 0.1M phosphate buffer, pH 8.5) with low-speedshaking for 1.5 h on titre-plate shaker at room temperature. The lysatecontaining plates were centrifuged at 4000 rpm and 4° C. for 10 min andthe clear lysate supernatant used for assay reactions. A 40 μL volume ofthe clear lysate was added to an assay reaction mixture of thesubstrates cyclohexanone, and norvaline, and the cofactor NADH, in 0.1Mphosphate buffer, pH 8.5. The resulting assay reaction mixture was 300μL in volume in a 96-well plate format and included 20 mM cyclohexanone,20 mM L-norvaline, and 5 mM NADH. These reaction mixture plates wereshaken overnight at high-speed on a titre-plate shaker at roomtemperature. Each reaction mixture was quenched with 300 μL CH₃CN anddiluted 10 fold in CH₃CN/H₂O/formic acid (50/50/0.1). The quenched anddiluted reaction mixtures were analyzed by LC-MS in multiple reactionmonitoring (MRM) mode. The relevant MS parameters were: [M + H]+: 200;Main fragment ions at CE = 20 ev: 154, 118, 83, 72, 55. The MRMtransitions used for monitoring product formation: 200/118; 200/72.³pyruvate/butylamine assay conditions: E. coli cells expressing thepolypeptide variant gene of interest were pelleted, placed in 96-wellplates and lysed in 250 μL lysis buffer (1 g/L lysozyme and 1 g/L PMBSin 0.1M phosphate buffer, pH 8.5) with low-speed shaking for 1.5 h ontitre-plate shaker at room temperature. The lysate containing plateswere centrifuged at 4000 rpm and 4° C. for 10 min and the clear lysatesupernatant used for assay reactions. A 20 μL volume of the clear lysatewas added to an assay mixture of the substrates pyruvate, butylamine,and the cofactor NADH, in 0.1M phosphate buffer, pH 8.5. The resultingassay reaction mixture was 300 μL in volume in a 96-well plate formatand included 20 mM pyruvate, 20 mM butylamine, and 5 mM NADH. Thereaction mixture plates were shaken overnight at high-speed on atitre-plate shaker at room temperature. Each reaction mixture wasquenched with 300 μL CH₃CN and diluted 10 fold in CH₃CN/H₂O/formic acid(50/50/0.1). The quenched and diluted reaction mixtures were analyzed byLC-MS in multiple reaction monitoring (MRM) mode. The relevant MSparameters were: [M + H]+: 146; Main fragment ion at CE = 20 ev: 100.MRM transitions used for monitoring product formation: 146/100.

TABLE 3B Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ ID Cyclohexanone/butylamine Assay¹ NO: Amino AcidDifferences Activity Ranking² (nt/aa) (relative to SEQ ID NO: 2) pH 8.5pH 10 87/88 A111M; K156T; N198H; Y259M; 1 1 Y280L; R292V; Y293H; 89/90K156T; V197P; N198H; Y259H; Y280L; 4 n.d.a³ R292P; Y293H; 91/92 A111M;S136G; K156S; V197I; N198H; 5 n.d.a³ M201L; Y259H; Y280L; R292V; Y293H;93/94 V197I; N198E; Y259M; Y280L; n.d.a³ 4 95/96 K156T; V197I; N198E;M201L; Y259H; 3 3 Y280L; R292V; Y293H; 97/98 A111M; S136G; N198H; Y259M;n.d.a³ 2 Y280L; R292S; Y293H;  99/100 K156V; V197P; N198E; M201L; 2n.d.a³ Y259M; Y280L; R292T; ¹ E. coli cells expressing the polypeptidevariant gene of interest were pelleted, placed in 96-well plates andlysed in 200 μL lysis buffer (1 g/L lysozyme and 1 g/L PMBS in 0.1Mphosphate buffer, pH 8.5) with low-speed shaking for 1.5 h ontitre-plate shaker at room temperature. The lysate containing plateswere centrifuged at 4000 rpm and 4° C. for 20 min and the clear lysatesupernatant used for assay reactions. A 200 μL volume of the clearlysate was added to an assay mixture of the substrates cyclohexanone,butylamine, and the cofactor NADH, in 0.1M phosphate buffer, pH 8.5 orpH 10. The resulting assay reaction mixture was 300 μL in volume in a96-well plate format and included 20 mM cyclohexanone, 20 mM butylamine,and 5 mM NADH. The reaction mixture plates were shaken overnight athigh-speed on a titre-plate shaker at room temperature. Each reactionmixture was quenched with 300 μL CH₃CN and diluted 10 fold inCH₃CN/H₂O/formic acid (50/50/0.1). The quenched and diluted reactionmixtures were analyzed by LC-MS in multiple reaction monitoring (MRM)mode. The relevant MS parameters were: [M + H]+: 156; Main fragment ionsat CE = 20 ev; 83, 74, 55. MRM transitions used for monitoring productformation: 156/83; 156/74; 156/55. ²The wild-type reference polypeptideof SEQ ID NO: 2 had no detectable activity with these substrates.Additionally, none of variant polypeptides of Table 3A had detectableactivity with these substrates. Thus, for each of the variantpolypeptides of SEQ ID NO: 88-100, the activity was assayed at theindicated pH as amount of product detected by LC-MS in the quenchedassay reaction mixture and was ranked with “1” indicating greatestactivity and “5” indicating least activity. ³n.d.a. = no detectableactivity

TABLE 3C Engineered Polypeptides and Relative Activity ImprovementsUsing SEP Enzyme Preparations Increased Activity¹ Increased Activity¹Diastereo- SEQ Amino Acid cyclohexanone/ cyclopentanone/ selectivity IDDifferences L-norvaline² L-norvaline³ Activity Ranking (major/minor) NO:(relative to (relative to (relative to acetophenone/ acetophenone/(nt/aa) SEQ ID NO: 2) SEQ ID NO: 2) SEQ ID NO: 2) L-norvaline⁴L-norvaline⁴ 3/4 N198A; 2.8 not tested not tested not tested 5/6 N198H;8.4   6.8 3 11.5   9/10 V197I; 1.7 not tested not tested not tested15/16 N198P; 2.2 not tested not tested not tested 71/72 R292Y; 6.5 13 24.7 73/74 R292S; 13 13 1 4.2 SFP assay reaction initial concentrations:ketone substrate: 50 mM; L-norvaline: 50 mM; engineered polypeptide SFP:5-10 g/L; NAD: 4.5 mM (3 g/L); sodium formate: 100 mM; formatedehydrogenase (FDH-101): 1 g/L; Buffer: 100 mM potassium phosphate, pH8.5. SFP assay reaction protocol: To 15 mL vial: (1) Add 0.29 mL of 100mM potassium phosphate buffer pH 8.5; (2) Add 0.2 mL of 1M sodiumformate in 100 mM potassium phosphate buffer, pH 8.5; (3) Add 0.2 mL ofNAD 30 g/L in 100 mM potassium phosphate buffer, pH 8.5; (4) Add 0.1 mLof FDH 20 g/L in 100 mM potassium phosphate buffer, pH 8.5; (5) Add10-20 mg engineered polypeptide SFP dissolved in 1 mL of 100 mMpotassium phosphate buffer, pH 8.5; (6) Add 0.2 mL of 0.5M norvaline in100 mM potassium phosphate buffer, pH 8.5; (7) Add appropriate volume ofketone substrate to get 50 mM final concentration (e.g., 12 μLcyclohexanone); (8) Adjust to pH 8.5 with 1M NaOH; (9) Stir at 900 RPMat 30° C. Final reaction volume: 2 mL. ³n.d.a. = no detectable activity

TABLE 3D Relative Activity Improvements for Various Unactivated KetoneSubstrates Using SFP Enzyme Preparations of Engineered PolypeptidesCyclohexanone/ Cyclohexanone/ Cyclohexanone/ SEQ butylamine methylamineaniline ID Assay¹ Assay¹ Assay¹ NO: Amino Acid Differences ActivityActivity Activity (nt/aa) (relative to SEQ ID NO: 2) Ranking² Ranking²Ranking² 87/88 A111M; K156T; N198H; Y259M; 1 3 3 Y280L; R292V; Y293H;89/90 K156T; V197P; N198H; Y259H; 3 4 2 Y280L; R292P; Y293H; 91/92A111M; S136G; K156S; V197I; n.d.a³ n.d.a³ not tested N198H; M201L;Y259H; Y280L; R292V; Y293H; 93/94 V197I; N198E; Y259M; Y280L; n.d.a³n.d.a³ not tested 95/96 K156T; V197I; N198E; M201L; 2 1 1 Y259H; Y280L;R292V; Y293H; 97/98 A111M; S136G; N198H; Y259M; n.d.a³ 5 4 Y280L; R292S;Y293H;  99/100 K156V; V197P; N198E; M201L; 1 2 n.d.a³ Y259M; Y280L;R292T; SFP assay reaction initial concentrations: ketone substrate: 50mM; amine substrate: 50 mM; engineered polypeptide SFP: 20-50 g/L; NAD:4.5 mM (3 g/L); sodium formate: 100 mM; formate dehydrogenase (FDH-101;commercially available from Codexis, Inc. Redwood City, California,USA): 1 g/L; Buffer: 100 mM potassium phosphate, pH 8.5. SFP assayreaction protocol: To 15 mL vial: (1) Add 0.29 mL of 100 mM potassiumphosphate buffer pH 8.5 (NOTE: if more than 20 g/L enzyme is used, 1.29mL buffer is added and the enzyme SFP is added to the reaction mixtureas a solid); (2) Add 0.2 mL of 1M sodium formate in 100 mM potassiumphosphate buffer, pH 8.5; (3) Add 0.2 mL of NAD 30 g/L in 100 mMpotassium phosphate buffer, pH 8.5; (4) Add 0.1 mL of FDH 20 g/L in 100mM potassium phosphate buffer, pH 8.5; (5) Add 40-100 mg enzyme SFPdissolved in 1 mL of 100 mM potassium phosphate buffer, pH 8.5; (6) Add0.2 mL of 0.5M norvaline in 100 mM potassium phosphate buffer, pH 8.5;(7) Add appropriate volume of ketone substrate to get 50 mM finalconcentration (e.g., 12 μL cyclohexanone); (8) Adjust to pH 8.5 with 1MNaOH; (9) Stir at 900 RPM at 30° C. Final reaction volume: 2 mL. ²Thewild-type reference polypeptide of SEQ ID NO: 2 had no detectableactivity with these substrates. Additionally, none of variantpolypeptides of Table 3A had detectable activity with these substrates.Thus, for each of the variant polypeptides of SEQ ID NO: 88-100, theactivity was assayed at the indicated pH as amount of product detectedby LC-MS in the quenched assay reaction mixture and was ranked with “1”indicating greatest activity and “5” indicating least activity. ³n.d.a.= no detectable activity

TABLE 3E Relative Activity Improvements for Various Unactivated KetoneSubstrates Using SFP Enzyme Preparations of Engineered Polypeptides2-Methoxy-cyclohexanone/ butylamine Assay¹ Cyclopentanone/Hydroxyacetone/ SEQ Amino Acid Diastereo- butylamine dimethylamine IDDifferences selectivity Assay¹ Assay¹ NO: (relative to Activity(major/minor) Activity Activity (nt/aa) SEQ ID NO: 2) Ranking² RankingRanking² Ranking² 87/88 A111M; K156T; N198H; 2 1 3 4 Y259M; Y280L;R292V; Y293H; 89/90 K156T; V197P; N198H; 3 1 4 not tested Y259H; Y280L;R292P; Y293H; 91/92 A111M; S136G; K156S; n.d.a³ n.d.a³ n.d.a³ not testedV197I; N198H; M201L; Y259H; Y280L; R292V; Y293H; 93/94 V197I; N198E;Y259M; n.d.a³ n.d.a³ 4 not tested Y280L; 95/96 K156T; V197I; N198E; 3 32 not tested M201L; Y259H; Y280L; R292V; Y293H; 97/98 A111M; S136G;N198H; 1 2 4 not tested Y259M; Y280L; R292S; Y293H;  99/100 K156V;V197P; N198E; 4 1 1 2 M201L; Y259M; Y280L; R292T; ¹SFP assay reactioninitial concentrations: ketone substrate: 50 mM; amine substrate: 50 mM;engineered polypeptide SFP: 20-50 g/L; NAD: 4.5 mM (3 g/L); sodiumformate: 100 mM; formate dehydrogenase (FDH-101; commercially availablefrom Codexis, Inc. Redwood City, California, USA): 1 g/L; Buffer: 100 mMpotassium phosphate, pH 8.5. SFP assay reaction protocol: To 15 mL vial:(1) Add 0.29 mL of 100 mM potassium phosphate buffer pH 8.5 (NOTE: ifmore than 20 g/L enzyme is used, 1.29 mL buffer is added and the enzymeSFP is added to the reaction mixture as a solid); (2) Add 0.2 mL of 1Msodium formate in 100 mM potassium phosphate buffer, pH 8.5; (3) Add 0.2mL of NAD 30 g/L in 100 mM potassium phosphate buffer, pH 8.5; (4) Add0.1 mL of FDH 20 g/L in 100 mM potassium phosphate buffer, pH 8.5; (5)Add 40-100 mg enzyme SFP dissolved in 1 mL of 100 mM potassium phosphatebuffer, pH 8.5; (6) Add 0.2 mL of 0.5M norvaline in 100 mM potassiumphosphate buffer, pH 8.5; (7) Add appropriate volume of ketone substrateto get 50 mM final concentration (e.g., 12 μL cyclohexanone); (8) Adjustto pH 8.5 with 1M NaOH; (9) Stir at 900 RPM at 30° C. Final reactionvolume: 2 mL. ²The wild-type reference polypeptide of SEQ ID NO: 2 hadno detectable activity with these substrates. Additionally, none ofvariant polypeptides of Table 3A had detectable activity with thesesubstrates. Thus, for each of the variant polypeptides of SEQ ID NO:88-100, the activity was assayed at the indicated pH as amount ofproduct detected by LC-MS in the quenched assay reaction mixture and wasranked with “1” indicating greatest activity and “5” indicating leastactivity. Variants with the same activity ranking were given the samenumber. ³n.d.a. = no detectable activity

TABLE 3F Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ Increased Increased ID Activity¹ Activity¹ NO:Amino Acid Differences (compound (compound (nt/aa) (Relative to SEQ IDNO: 96) (1i) assay²) (1j) assay³) 111/112 A284M; ++++ ++ 113/114 D324T;++ + 115/116 D160N; ++++ + 117/118 V184R; ++ + 119/120 V186R; +++ +121/122 G157L; ++ − 123/124 G157N; ++ + 125/126 S232A; + + 127/128N288G; + − 129/130 Q220D; ++ + 131/132 A284L; + + 133/134 L223T; ++ +135/136 E261A; ++ + 137/138 A243G; F294V; ++ − 139/140 N288S; ++ ++141/142 G157M; ++ + 143/144 I287T; ++ ++ 145/146 V184K; ++ + 147/148A284Y; + − 149/150 E261K; +++ + 151/152 E261S; ++ + 153/154 E261I; ++ +155/156 A158K; + + 157/158 D324L; ++ ++ 159/160 E261T; +++ + 161/162E261G; I287T; ++ ++ 163/164 Q149L; ++ − 165/166 S178E; ++ − 167/168K260G; ++ − 169/170 V184Q; + + 171/172 A284K; ++ − 173/174 G157D;K246W; + − 175/176 S29R; + + 177/178 S100W; ++ − 179/180 S356R; ++ +181/182 S67D; ++ − 183/184 S232R; ++ + 185/186 I287S; + + 187/188T332V; + + 189/190 V186K; ++ + 191/192 E261R; +++ ++ 193/194 G157R; ++ +195/196 S67A; S232R; + + 197/198 A311V; ++ + 199/200 G353E; + + 201/202F295R; + − 203/204 S29R; V184R; L223T; E261A; I287T; N288S; n.d. +++++205/206 S29R; S67D; V186R; L223T; E261R; N288S; n.d. ++++ 207/208 S29R;S67D; G157R; V186R; Q220D; L223T; E261A; N288S; n.d. +++++ 209/210 S67D;V184R; L223T; E261R; I287T; N288G; A311V; n.d. +++ 211/212 S29R; S67D;G157R; V184R; V186K; A284M; N288S; D324T; n.d. +++ 213/214 S29R; G157R;D160N; V186K; Q220D; L223T; A284M; I287T; n.d. ++++ 215/216 S29R; G157R;E261K; I287T; n.d. ++++ 217/218 S67D; G157R; V186K; Q220D; I287T; N288G;A311V; n.d. +++ 219/220 S29R; G157R; V184R; Q220D; E261K; N288S; A311V;n.d. ++++ 221/222 S67D; V184R; V186K; L223T; E261A; A284M; N288S; n.d.+++ 223/224 S29R; S67D; G157R; V186R; L223T; A284M; I287T; N288G; n.d.++++ 225/226 S29R; L223T; E261K; I287T; A311V; n.d. +++++ 227/228 S29R;S67D; D160N; V186K; E261R; A284M; n.d. ++ 229/230 S67D; G157R; A185V;V186K; Q220D; E261K; I287T; N288S; n.d. ++++ A311V; 231/232 S29R; S67D;G157R; V184R; V186R; E261A; I287T; N288G; n.d. ++ 233/234 S29R; S67D;G157R; L223T; E261K; N288S; A311V; D324T; n.d. +++++ 235/236 S29R; S67D;G157R; Q220D; L223T; E261K; I287T; N288S; A311V; n.d. +++++ D324T;237/238 S29R; S67D; D160N; V186K; Q220D; E261A; I287T; N288S; D324T;n.d. +++ 239/240 S29R; S67D; G157R; E261R; I287T; N288S; n.d. +++++241/242 S67D; D160N; V186K; E261A; A284M; N288S; A311V; D324T; n.d. +243/244 S29R; V186K; Q220D; L223T; E261S; I287T; N288G; A311V; n.d. ++++D324T; 245/246 S29R; S67D; V184R; Q220D; E261A; A284M; N288G; A311V;n.d. ++++ D324T; 247/248 S67D; G157R; V184R; V186R; Q220D; L223T; A284M;N288S; n.d. ++++ 249/250 S67D; G157R; D160N; V184R; L223T; E261S; I287T;n.d. ++++ 251/252 S29R; V184R; L223T; E261S; A284M; I287T; n.d. +++++253/254 S67D; G157R; V184R; V186K; L223T; E261A; I287T; n.d. ++++255/256 S67D; G157R; V184R; V186R; L223T; E261R; N288S; A311V; n.d. +++D324T; 257/258 V184R; Q220D; E261K; I287T; N288S; n.d. ++++ 259/260S29R; S67D; G157R; D160N; V186K; E261R; I287T; A311V; n.d. +++ 261/262S67D; V186K; L223T; E261S; A284M; I287T; N288G; A311V; n.d. +++ D324T;263/264 S29R; L223T; I287T; N288S; A311V; D324T; n.d. +++++ 265/266S29R; S67D; G157R; V184R; L223T; E261S; I287T; N288S; n.d. +++++ 267/268S29R; S67D; G157R; D160N; V186K; L223T; E261K; A284M; n.d. +++ N288S;A311V; D324T; 269/270 S29R; S67D; G157R; V186K; L223T; E261A; I287T;N288S; A311V; n.d. ++++ 271/272 S67D; G157R; D160N; V186R; L223T; E261S;A284M; I287T; n.d. ++ N288G; A311V; 273/274 S67D; D160N; V186R; L223T;A284M; N288G; n.d. ++ 275/276 S67D; G157R; L223T; E261A; I287T; D324T;n.d. ++++ 277/278 S29R; S67D; G157R; V184R; L223T; E261R; A284M; N288S;n.d. ++++ 279/280 S67D; G157R; V184R; V186R; Q220D; E261S; I287T; N288G;n.d. +++ 281/282 S67D; G157R; V186K; E261A; I287T; N288S; D324T; n.d.++++ 283/284 S67D; G157R; V184R; Q220D; N288S; A311V; n.d. ++++ 285/286S67D; E261S; N288S; n.d. ++ 287/288 S29R; G157R; D160N; V186K; Q220D;E261S; I287T; n.d. ++++ 289/290 S29R; S67D; G157R; V186R; E261A; I287T;N288S; n.d. ++++ 291/292 S29R; G157R; D160N; V186K; I287T; N288S; n.d.++++ ¹Levels of increased activity were determined relative to thereference polypeptide of SEQ ID NO: 96 and defined as follows: “−” =activity less than or equal to reference polypeptide; “+” = at least1.1-fold but less than 2.5-fold increased activity; “++” = at least2.5-fold but less than 5-fold increased activity; “+++” = at least5-fold increased activity but less than 10-fold; “++++” = at least 10fold but less than 15-fold; and “+++++” at least 15-fold but less than20-fold. ²Substrate compound (1i) activity assay: Enzyme lysatepreparation: E. coli cells expressing the polypeptide variant gene ofinterest were pelleted, placed in 96-well plates and lysed in 150 μLlysis buffer (1 g/L lysozyme and 1 g/L PMBS in 0.1M phosphate buffer, pH8.5) with low-speed shaking for 1.5 h on titre-plate shaker at roomtemperature. The lysate containing plates were centrifuged at 4000 rpmand 4° C. for 10 min and the clear lysate supernatant used for assayreactions. HTP assay reaction: The enzyme assay reaction was carried outin a total volume of 250 μL in a 96-well plate format. The assayreaction was initiated by adding the following to each well containing150 μL of the lysate: (i) 37.5 μL of FDH cofactor recycling pre-mix(pre-mix contains 666.7 mM sodium formate, 20 g/L NADH, 6.66 g/LFDH-101); (ii) 50 μL of butylamine stock solution (500 mM); and 12.5 μLketone substrate stock solution (1M compound (1i) in DMSO). Theresulting assay reaction included 50 mM ketone substrate compound (1i),100 mM amine substrate butylamine (compound (2b)), 100 mM sodiumformate, 3 g/L NADH, 1 g/L FDH-101, 100 mM potassium phosphate, pH 8.5,5% (v/v) DMSO. The reaction plate was heat-sealed and shaken at 250 rpmovernight (20-24 h) at 35° C. Work-up and analysis: Each reactionmixture was quenched by adding 250 μL CH₃CN, shaken, and centrifuged at4000 rpm and 4° C. for 10 min. 20 μL of the quenched mixture was diluted10 fold in 180 μL CH₃CN/H₂O (50/50) with mixing. 10 μL of this 10-folddilution mixture was then further diluted in 190 μL CH₃CN/H₂O (50/50)for a total 400 fold diluted mixtures. These mixtures then were analyzedfor product compound (3n) formation by LC-MS in MRM mode as described inExample 4. ³Substrate compound (1j) activity assay: Enzyme lysatepreparation: E. coli cells expressing the polypeptide variant gene ofinterest were pelleted, placed in 96-well plates and lysed in 250 μLlysis buffer (1 g/L lysozyme and 1 g/L PMBS in 0.1M phosphate buffer, pH8.5, 50 mM triethanol amine, 100 mM potassium chloride) with low-speedshaking for 1.5 h on titre-plate shaker at room temperature. The lysatecontaining plates were centrifuged at 4000 rpm and 4° C. for 10 min andthe clear lysate supernatant used for assay reactions. HTP assayreaction: The enzyme assay reaction was carried out in a total volume of250 μL in a 96-well plate format. The assay reaction was initiated byadding the following to each well containing 150 μL of the lysate: (i)37.5 μL of FDH cofactor recycling pre-mix (pre-mix contains 666.7 mMsodium formate, 20 g/L NAD, 6.66 g/L FDH-101); (ii) 50 μL of butylaminestock solution (500 mM in 100 mM potassium phosphate, 50 mM triethylamine, 100 mM potassium chloride, pH 8.5); and 12.5 μL ketone substratestock solution (1M compound (1j) in DMSO). The resulting assay reactionincluded 50 mM ketone substrate compound (1i), 100 mM amine substratebutylamine (compound (2b)), 100 mM sodium formate, 3 g/L NAD, 1 g/LFDH-101, 50 mM triethyl amine, 100 mM potassium chloride, 100 mMpotassium phosphate, pH 8.5, 5% (v/v) DMSO. The reaction plate washeat-sealed and shaken at 250 rpm overnight (20-24 h) at 35° C. Work-upand analysis: Each reaction mixture was quenched by adding 250 μL CH₃CN,shaken, and centrifuged at 4000 rpm and 4° C. for 10 min. 20 μL of thequenched mixture was diluted 10 fold in 180 μL CH₃CN/H₂O (50/50) withmixing. 10 μL of this 10-fold dilution mixture was then further dilutedin 190 μL CH₃CN/H₂O (50/50) for a total 400 fold diluted mixtures. Thesemixtures then were analyzed for product compound (3o) formation by LC-MSin MRM mode as described in Example 4. “n.d.” = not determined

TABLE 3G Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ Increased ID Activity¹ NO: Amino Acid Differences(compound (nt/aa) (Relative to SEQ ID NO: 96) (1j) assay) 293/294 S29R;G157R; V184K; Q220H; L223T; S232A; E261S; A284L; I287T; N288S; ++²T332V; G353E; 295/296 S29R; S97P; G157R; A158K; V184R; Q220H; L223T;S232A; E261K; A284L; ++² I287T; D324L; T332V; G353E; 297/298 S29R;G157R; A158K; V184K; Q220D; L223T; S232R; E261R; A284M; I287T; +² N288S;299/300 S29R; V184R; Q220D; L223T; E261S; A284M; I287T; N288S; D324L;G353E; +² 301/302 S29R; V184Q; Q220H; L223T; S232R; E261R; A284M; I287T;N288S; T332V; ++² G353E; 303/304 S29R; G157R; V184K; Q220D; L223T;S232A; E261R; A284L; I287T; D324T; +² G353E; 305/306 S29R; G157R; V184Q;Q220H; L223T; S232R; E261I; A284L; I287T; N288S; +² 307/308 S29R; G157R;A158K; V184Q; Q220D; L223T; S232R; E261G; I287T; N288S; +² D324L; T332V;G353E; 309/310 S29R; V184K; Q220D; L223T; S232R; E261S; A284M; I287T;N288S; G353E; +² 311/312 S29R; G157R; A158K; V184Q; Q220H; L223T; E261R;A284M; I287T; N288S; ++² D324L; T332V; 313/314 S29R; V184K; Q220H;L223T; S232R; E261I; A284M; I287T; N288S; D324L; +² G353E; 315/316 S29R;G157R; V184Q; L223T; S232R; E261S; I287T; D324L; T332V; G353E; ++²317/318 S29R; G157R; V184R; L223T; S232A; E261S; A284M; I287T; N288S; +²319/320 S29R; G157R; A158K; V184K; Q220H; L223T; S232R; E261K; A284M;I287T; +² N288S; D324T; G353E; 321/322 S29R; V184Q; Q220H; L223T; S232R;E261K; A284L; I287T; N288S; D324L; +² G353E; 323/324 S29R; V184K; Q220H;L223T; S232R; E261S; A284M; I287T; N288S; D324T; +² T332V; G353E;325/326 S29R; G157R; A158K; V184Q; Q220D; L223T; S232A; E261I; I287T;N288S; +² 327/328 S29R; G157R; V184R; Q220D; L223T; S232A; E261I; A284M;I287T; N288S; +² 329/330 S29R; G157R; V184R; Q220H; L223T; S232R; E261K;A284L; I287T; N288S; +² 331/332 S29R; G157R; V184Q; Q220H; L223T; S232A;E261R; A284M; I287T; N288S; ++² G353E; 333/334 S29R; V184Q; L223T;S232R; E261R; A284L; I287T; T332V; G353E; +² 335/336 S29R; V184R; Q220D;L223T; E261I; I287T; N288S; D324T; T332V; G353E; +² 337/338 S29R; G157R;V184Q; Q220H; L223T; S232R; E261S; A284M; I287T; N288S; +² D324T;339/340 S29R; V184Q; Q220H; L223T; S232R; E261I; A284L; I287T; N288S;T332V; +² 341/342 S29R; G157R; V184K; Q220D; L223T; S232A; E261I; A284M;I287T; N288S; +² G353E; 343/344 S29R; G157R; V184K; L223T; S232R; E261S;A284L; I287T; N288S; D324T; +² G353E; 345/346 S29R; S97P; G157R; V184Q;Q220H; L223T; E261K; A284M; I287T; N288S; ++² G353E; 347/348 S29R;G157R; A158K; V184K; Q220D; L223T; S232R; E261I; I287T; N288S; ++²D324T; G353E; 349/350 S29R; G157R; V184Q; L223T; E261I; A284M; I287T;N288S; +² 351/352 S29R; V184Q; Q220H; L223T; S232R; E261R; A284M; I287T;N288S; +² 353/354 S29R; G157R; V184Q; Q220H; L223T; S232A; E261I; A284M;I287T; N288S; +++² D324L; T332V; G353E; 355/356 S29R; G157M; V184R;L223T; E261A; A284M; I287T; N288G; A311V; S328G; +² 357/358 S29R; S67A;G157L; V184R; L223T; E261A; A284M; I287T; +² 359/360 A20T; S29R; Q149L;G157H; V184R; L223T; E261S; A284M; I287T; A311V; +² 361/362 A20T; S29R;G157L; V184R; L223T; E261A; A284M; I287S; N288G; A311V; +² 363/364 A20T;S29R; Q149L; G157L; V184R; L223T; E261K; A284M; I287T; N288G; +² A311V;365/366 S29R; S67A; G157R; V184R; L223T; E261S; A284M; I287T; N288G;A311V; +² S356R; 367/368 S29R; Q149L; G157L; V184R; L223T; E261A; A284M;I287T; N288G; A311V; +² S356R; 369/370 A20T; S29R; S67A; Q149L; G157L;V184R; L223T; E261S; A284M; I287S; +² N288G; F294I; A311V; 371/372 S29R;S67A; G157R; V184R; L223T; E261A; A284Y; I287T; A311V; S328G; +² S356R;373/374 S29R; G157R; V184R; L223T; E261S; A284L; I287T; A311V; +²375/376 S29R; Q149L; G157L; V184R; L223T; E261S; A284M; I287T; N288G;A311V; +² S328G; 377/378 S29R; V184R; L223T; E261S; A284M; I287T; A311V;G353E; +² 379/380 S29R; V184R; L223T; E261S; A284M; I287T; A311V; +²381/382 S29R; G157L; V184R; L223T; E261S; A284M; I287T; +++³ 383/384S4L; S29R; V184R; L223T; E261S; A284M; I287T; ++³ 385/386 K5T; S29R;V184R; L223T; E261S; A284M; I287T; ++³ 387/388 S29R; V184R; L223T;E261S; A284M; I287T; S328A; +³ 389/390 S29R; V184R; L223T; E261S; A284M;I287T; D324L; +³ 391/392 S29R; V184R; L223T; E261S; A284M; I287T; L323T;+³ 393/394 S29R; V184R; L223T; E261S; A284M; I287T; S328G; +³ 395/396S29R; V184R; L223T; E261S; A284M; I287T; F294V; +³ 397/398 S29R; A154F;V184R; L223T; E261S; A284M; I287T; +³ 399/400 S4H; S29R; A158K; V184R;L223T; E261S; A284M; I287T; +³ 401/402 S4H; S29R; V184R; L223T; E261S;A284M; I287T; +³ 403/404 S29R; V184R; L223T; E261S; A284M; I287T; +³405/406 S29R; V184R; L223T; E261S; A284M; I287T; L323V; +³ 407/408 S29R;V184R; L223T; E261S; Q265G; A284M; I287T; +³ 409/410 S29R; G157R; V184R;L223T; E261S; A284M; I287T; +³ 411/412 S29R; Y183C; V184R; L223T; E261S;A284M; I287T; +³ 413/414 S29R; V184R; L223T; E261S; A284M; I287T; L308F;+³ 415/416 S29R; V184R; L223T; S232R; E261S; A284M; I287T; +³ 417/418S29R; A154Y; V184R; L223T; E261S; A284M; I287T; +³ 419/420 S29R; V184R;L223T; E261S; Q265Y; A284M; I287T; +³ 421/422 S29R; V184R; L223T; E261S;A284M; I287T; F294A; +³ 423/424 S29R; N94R; V184R; L223T; E261S; A284M;I287T; +³ 425/426 S29R; I155M; V184R; L223T; E261S; A284M; I287T; +³427/428 S29R; V184R; L223T; E261S; A284M; I287T; S328E; +³ 429/430 S29R;N94K; V184R; L223T; E261S; A284M; I287T; +³ 431/432 S29R; V184R; L223T;E261S; A284M; I287T; T332V; +³ 433/434 S29R; A154Q; V184R; L223T; E261S;A284M; I287T; +³ 435/436 S29T; V184R; L223T; E261S; A284M; I287T; +³437/438 S29R; V184R; L223T; C256V; E261S; A284M; I287T; +³ 439/440 S29R;V184R; L223T; E261S; Q265L; A284M; I287T; +³ 441/442 S29R; V184R; L223T;E261S; A284M; I287T; A311T; +³ ¹Levels of increased activity weredetermined relative to the activity of the reference polypeptide of SEQID NO: 252 and defined as follows: “+” = at least 1.3-fold but less than2-fold increased activity; “++” = at least 2-fold but less than 3-foldincreased activity; “+++” = at least 3-fold increased activity but lessthan 5-fold. ²Substrate compound (1j) activity assay: Enzyme lysatepreparation: E. coli cells expressing the polypeptide variant gene ofinterest were pelleted, placed in 96-well plates and lysed in 250 μLlysis buffer (1 g/L lysozyme and 1 g/L PMBS in 0.1M phosphate buffer, pH8.0) with low-speed shaking for 2 h on titre-plate shaker at roomtemperature. The lysate containing plates were centrifuged at 4000 rpmand 4° C. for 10 min and the clear lysate supernatant used for assayreactions. HTP assay reaction: The enzyme assay reaction was carried outin a total volume of 100 μL in a 96-well plate format. The assayreaction was initiated by adding 55 μL volume of the clarified lysate to45 μL of an assay mixture comprised of: (i) 50 mM ketone substratecompound (1j) (15 μL of 330 mM ketone substrate stock in DMSO); (ii) 100mM butylamine (20 μL of 500 mM butylamine stock); and (iii) NADHcofactor recycling system pre-mix (10 μL of a solution of 10 g/LGDH-105, 1M glucose, 30 g/L NAD in 0.1M potassium phosphate buffer, pH8.5). The resulting assay reaction included 50 mM compound (1j), 100 mMbutylamine, 100 mM glucose, 1 g/L glucose dehydrogenase GDH-105, 3 g/LNADH, in 0.1M potassium phosphate, pH 8.0, and 25% (v/v) DMSO. Thereaction mixture plates were shaken overnight (16-20 h) at 250 rpm on atitre-plate shaker at 44° C. Work-up and analysis: Each reaction mixturein the plate was quenched with 100 μL CH₃CN, heat-sealed, shaken, andcentrifuged at 4000 rpm, 4 C., for 10 min. 20 μL of the quenchedreaction was added to 180 uL of 50% acetonitrile with 0.1%trifluoroacetic acid. The diluted reaction mixtures then were analyzedfor product compound (3o) formation by LC-MS in MRM mode as described inExample 4.. ³Substrate compound (1j) activity assay: Lysate preparation,HTP assay reaction, and work-up and analysis were carried out asdescribed directly above (Note 2, Table 3G) except that 15% (v/v) DMSOwas present in the assay reaction.

TABLE 3H Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ Increased ID Activity¹ NO: Amino Acid Differences(compound (nt/aa) (Relative to SEQ ID NO: 96) (1j) assay²) 443/444 S29R;G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; I287T; N288S; +++E296L; D324L; T332V; G353E; 445/446 S29R; N94K; A154Y; G157R; Y183C;V184Q; Q220H; L223T; S232A; E261I; ++ Q265L; A284M; I287T; N288S; D324L;T332V; G353E; 447/448 S29R; G157R; V184Q; Q220H; L223T; S232A; H259V;E261I; A284M; I287T; ++ N288S; D324L; T332V; G353E; 449/450 N14P; S29R;G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; I287T; ++ N288S; D324L;T332V; G353E; 451/452 S29R; N94K; G157L; Y183C; V184Q; Q220H; L223T;S232A; E261I; A284M; ++ I287T; N288S; D324L; T332V; G353E; 453/454 S29R;N94K; A154Y; G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; ++ I287T;N288S; L323V; D324L; T332V; G353E; 455/456 S29R; A154Q; G157R; G177H;V184Q; Q220H; L223T; S232R; C256V; E261I; ++ A284M; I287T; N288S; A311V;L323C; D324L; S328E; T332V; G353E; 457/458 S29T; A37H; A154Q; G157R;V184Q; Q220H; L223T; S232R; E261I; A284M; ++ I287T; N288S; A311V; D324L;S328A; T332V; G353E; 459/460 S29R; G157R; V184Q; Q220H; L223T; S232A;E261I; A284M; I287T; N288S; ++ D324L; T332V; G353E; 461/462 S29R; G157R;V184Q; Q220H; L223T; S232A; E261I; A279Y; A284M; I287T; ++ N288S; D324L;T332V; G353E; 463/464 S29R; N94K; A154Y; G157L; V184Q; Q220H; L223T;S232A; E261I; Q265Y; ++ A284M; I287T; N288S; D324L; T332V; G353E;465/466 S29R; G157R; V184Q; Q220H; L223T; S232A; E261I; A279V; A284M;I287T; ++ N288S; D324L; T332V; G353E; 467/468 S29R; A154M; G157R; G177H;V184Q; Q220H; L223T; S232R; E261I; A284M; ++ I287T; N288S; A311V; D324L;T332V; G353E; 469/470 S4L; S29R; N94K; G157R; Y183C; V184Q; Q220H;L223T; S232A; E261I; ++ A284M; I287T; N288S; D324L; T332V; G353E;471/472 S29R; G157R; V184Q; Q220H; L223T; S232A; E261I; A279F; A284M;I287T; + N288S; D324L; T332V; G353E; 473/474 S29R; A111R; G157R; V184Q;Q220H; L223T; S232A; E261I; A284M; I287T; + N288S; D324L; T332V; G353E;475/476 S29R; N94K; G157R; Y183C; V184Q; Q220H; L223T; S232A; E261I;Q265L; + A284M; I287T; N288S; D324L; T332V; G353E; 477/478 S29R; E124L;A154F; I155M; G157R; V184Q; Q220H; L223T; S232R; E261I; + A284M; I287T;N288S; A311T; D324L; T332V; G353E; 479/480 S29R; A37H; E124N; A154F;I155M; G157R; G177H; V184Q; Q220H; L223T; + S232A; E261I; A284M; I287T;N288S; A311V; L323C; D324L; S328E; T332V; G353E; 481/482 S29R; E124N;I155M; G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; + I287T; N288S;D324L; S328E; T332V; G353E; 483/484 S29R; N94K; G157R; Y183C; V184Q;Q220H; L223T; S232A; E261I; Q265L; + A284M; I287T; N288S; F294A; L308F;D324L; T332V; G353E; 485/486 S29R; N94K; G157L; V184Q; Q220H; L223T;S232A; E261I; Q265Y; A284M; + I287T; N288S; D324L; T332V; G353E; 487/488S29R; N94R; G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; I287T; +N288S; D324L; T332V; G353E; 489/490 S29R; N94K; G157L; Y183C; V184Q;Q220H; L223T; S232A; E261I; Q265G; + A284M; I287T; N288S; D324L; T332V;G353E; 491/492 S29R; E124L; I155M; G157R; V184Q; Q220H; L223T; S232A;E261I; A284M; + I287T; N288S; A311T; D324L; T332V; G353E; 493/494 S29R;G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; I287T; N288S; + D297A;D324L; T332V; G353E; 495/496 S29R; A154Q; G157R; V184Q; Q220H; L223T;S232A; E261I; A284M; I287T; + N288S; A311T; D324L; T332V; G353E; 497/498S29R; A154F; I155M; G157R; G177H; V184Q; Q220H; L223T; S232A; E261I; +A284M; I287T; N288S; D324L; T332V; G353E; 499/500 S29R; A154F; G157R;V184Q; Q220H; L223T; S232R; E261I; A284M; I287T; + N288S; A311C; D324L;T332V; G353E; 501/502 S29R; A37H; A154F; I155M; G157R; V184Q; Q220H;L223T; S232R; C256V; + E261I; A284M; I287T; N288S; A311V; D324L; S328G;T332V; G353E; 503/504 S29R; N94K; G157M; V184Q; Q220H; L223T; S232A;E261I; Q265L; A284M; + I287T; N288S; L308F; D324L; T332V; G353E; 505/506S29R; A37H; E124L; A154Q; I155M; G157R; V184Q; Q220H; L223T; S232A; +E261I; A284M; I287T; N288S; A311V; D324L; T332V; G353E; 507/508 S29R;A154M; G157R; V184Q; Q220H; L223T; S232R; C256V; E261I; A284M; + I287T;N288S; A311V; D324L; S328E; T332V; G353E; 509/510 S29R; G157R; V184Q;Q220H; L223T; S232A; W258D; E261I; A284M; I287T; + N288S; D324L; T332V;G353E; 511/512 S29R; N94K; G157M; V184Q; Q220H; L223T; S232A; E261I;Q265L; A284M; + I287T; N288S; D324L; T332V; G353E; 513/514 S29R; N94R;G157R; Y183C; V184Q; Q220H; L223T; S232A; E261I; Q265G; + A284M; I287T;N288S; D324L; T332V; G353E; 515/516 S29T; G157R; V184Q; Q220H; L223T;S232A; E261I; A284M; I287T; N288S; + D324L; T332V; G353E; 517/518 S29R;G157R; V184Q; Q220H; L223T; I226L; S232A; E261I; A284M; I287T; + N288S;D324L; T332V; G353E; 519/520 S29R; I155M; G157R; G177H; V184Q; Q220H;L223T; S232A; E261I; A284M; + I287T; N288S; A311V; L323M; D324L; S328E;T332V; G353E; 521/522 S29T; A37H; A154Q; G157R; V184Q; Q220H; L223T;S232A; E261I; A284M; + I287T; N288S; A311C; D324L; S328E; T332V; G353E;523/524 S29R; N94K; G157R; Y183C; V184Q; Q220H; L223T; S232A; E261I;A284M; + I287T; N288S; F294V; D324L; T332V; G353E; 525/526 S29R; N94K;A154F; G157M; Y183C; V184Q; Q220H; L223T; S232A; E261I; + A284M; I287T;N288S; D324L; T332V; G353E; 527/528 S29R; N94R; G157L; V184Q; Q220H;L223T; S232A; E261I; Q265L; A284M; + I287T; N288S; L323V; D324L; T332V;G353E; 529/530 S29R; N94R; G157L; V184Q; Q220H; L223T; S232A; E261I;Q265L; A284M; + I287T; N288S; L308F; D324L; T332V; G353E; 531/532 S29R;A154M; G157R; G177H; V184Q; Q220H; L223T; S232R; E261I; A284M; + I287T;N288S; A311C; D324L; S328E; T332V; G353E; 533/534 S29R; A154M; G157R;G177H; V184Q; Q220H; L223T; S232A; C256V; E261I; + A284M; I287T; N288S;A311V; D324L; T332V; G353E; 535/536 S29R; A154F; G157R; V184Q; Q220H;L223T; S232A; E261I; A284M; I287T; + N288S; D324L; S328A; T332V; G353E;537/538 S29R; G157L; V184Q; Q220H; L223T; S232A; E261I; Q265L; A284M;I287T; + N288S; D324L; T332V; G353E; 539/540 S29R; G157R; Y183C; V184Q;Q220H; L223T; S232A; E261I; A284M; I287T; + N288S; L323V; D324L; T332V;G353E; 541/542 S4R; S29R; K74R; G157R; C163T; V184Q; Q220H; L223T;S232A; E261I; + A284M; I287T; N288S; D324L; T332V; G353E; 543/544 S29R;A154M; G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; I287T; + N288S;D324L; S328E; T332V; G353E; 545/546 S29R; A154M; I155M; G157R; V184Q;Q220H; L223T; S232A; C256V; E261I; + A284M; I287T; N288S; A311V; D324L;S328E; T332V; G353E; 547/548 S4H; S29R; N94R; A154Y; G157L; V184Q;Q220H; L223T; S232A; E261I; + A284M; I287T; N288S; L323T; D324L; T332V;G353E; 549/550 S29R; G157R; Y183C; V184Q; Q220H; L223T; S232A; E261I;Q265L; A284M; + I287T; N288S; F294V; D324L; T332V; G353E; 551/552 S29R;A154F; G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; I287T; + N288S;A311C; D324L; T332V; G353E; 553/554 S29R; A37H; E124L; A154F; G157R;G177H; V184Q; Q220H; L223T; S232A; + E261I; A284M; I287T; N288S; A311C;L323M; D324L; S328E; T332V; G353E; 555/556 S29R; A154Y; G157R; Y183C;V184Q; Q220H; L223T; S232A; E261I; Q265L; + A284M; I287T; N288S; D324L;T332V; G353E; 557/558 S29R; N94T; G157R; V184Q; Q220H; L223T; S232A;E261I; Q265L; A284M; + I287T; N288S; D324L; T332V; G353E; 559/560 S29R;G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; I287T; N288S; + L323I;D324L; T332V; G353E; 561/562 S29R; A154F; I155M; G157R; V184Q; Q220H;L223T; S232R; C256V; E261I; + A284M; I287T; N288S; A311V; D324L; T332V;G353E; 563/564 S29R; A154Q; G157R; V184Q; Q220H; L223T; S232A; E261I;A284M; I287T; + N288S; A311V; D324L; T332V; G353E; 565/566 S29R; E124N;A154Q; G157R; G177C; V184Q; Q220H; L223T; S232A; E261I; + A284M; I287T;N288S; D324L; S328A; T332V; G353E; 567/568 S29R; A154Q; G157R; V184Q;Q220H; L223T; S232A; E261I; A284M; I287T; + N288S; D324L; S328E; T332V;G353E; 569/570 S29R; N94T; G157R; V184Q; Q220H; L223T; S232A; E261I;Q265L; A284M; + I287T; N288S; L308F; D324L; T332V; G353E; 571/572 S29R;G157R; V184Q; Q220H; L223T; S232A; E261I; A284M; I287T; N288S; + E296W;D324L; T332V; G353E; 573/574 S29R; G157R; V184Q; Q220H; L223T; S232A;E261I; I270G; A284M; I287T; + N288S; D324L; T332V; G353E; 575/576 S29R;N94K; G157R; Y183C; V184Q; Q220H; L223T; S232A; E261I; Q265G; + A284M;I287T; N288S; F294A; D324L; T332V; G353E; ¹Levels of increased activitywere determined relative to the activity of the reference polypeptide ofSEQ ID NO: 354 and defined as follows: “+” = at least 1.3-fold but lessthan 2-fold increased activity; “++” = at least 2-fold but less than3-fold increased activity; “+++” = at least 3-fold increased activitybut less than 5-fold. ²Substrate compound (1j) activity assay: Lysatepreparation, HTP assay reaction, and work-up and analysis were carriedout as described in Note 3, Table 3G.

TABLE 3I Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ Increased ID Activity¹ NO: Amino Acid Differences(compound (nt/aa) (Relative to SEQ ID NO: 96) (1j) assay) 577/578 S29R;N94R; G157R; V184Q; Q220H; L223T; S232A; H259V; E261I; A279F; ++ A284M;I287T; N288S; D324L; T332V; G353E; 579/580 S29R; N94K; A111R; A154Y;G157R; V184Q; Q220H; L223T; S232A; H259V; + E261I; A279V; A284M; I287T;N288S; D324L; T332V; G353E; 581/582 S29R; S137N; G157R; V184Q; Q220H;L223T; S232A; H259V; E261I; A279V; + A284M; I287T; N288S; D324L; T332V;G353E; 583/584 S29R; N94K; A111R; G157R; V184Q; Q220H; L223T; S232A;H259V; E261I; + A279F; A284M; I287T; N288S; D324L; T332V; G353E; 585/586S29R; N94R; G157R; V184Q; Q220H; L223T; S232A; H259V; E261I; A279V; ++A284M; I287T; N288S; D324L; T332V; G353E; 587/588 S29R; N94R; A154Y;G157R; V184Q; Q220H; L223T; S232A; H259V; E261I; ++ A279F; A284M; I287T;N288S; E296W; D324L; T332V; G353E; 589/590 S29R; N94R; A111R; G157R;V184Q; Q220H; L223T; S232A; H259V; E261I; ++ A279V; A284M; I287T; N288S;D324L; T332V; G353E; 591/592 S29R; N94K; G157R; V184Q; Q220H; L223T;S232A; H259V; E261I; A279F; ++ A284M; I287T; N288S; D324L; T332V; G353E;593/594 S29R; N94R; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A;H259V; +++ E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E;595/596 S29R; N94K; G157R; V184Q; Q220H; L223T; S232A; H259V; E261I;A279F; ++ A284M; I287T; N288S; E296W; D324L; T332V; G353E; 597/598 S29R;N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; H259V; +++ E261I;A279V; A284M; I287T; N288S; D324L; T332V; G353E; 599/600 S29R; N94K;G157R; V184Q; Q220H; L223T; S232A; H259V; E261I; A284M; + I287T; N288S;E296W; D324L; T332V; G353E; 601/602 S29R; N94K; G157R; V184Q; Q220H;L223T; S232A; H259V; E261I; A279V; +++ A284M; I287T; N288S; D324L;T332V; G353E; 603/604 S29R; N94R; S137N; G157R; V184Q; Q220H; L223T;S232A; H259V; E261I; + A279F; A284M; I287T; N288S; D324L; T332V; G353E;605/606 S29R; G157R; V184Q; Q220H; L223T; S232A; H259V; E261I; A284M;I287T; + N288S; E296W; D324L; T332V; G353E; 607/608 S29R; N94R; A111R;S137N; G157R; V184Q; Q220H; L223T; S232A; H259V; ++ E261I; A279F; A284M;I287T; N288S; D324L; T332V; G353E; 609/610 S29R; A154Y; G157R; V184Q;Q220H; L223T; S232A; H259V; E261I; A279F; ++ A284M; I287T; N288S; E296W;D324L; T332V; G353E; 611/612 S29R; G157R; V184Q; Q220H; L223T; S232A;H259V; E261I; A279F; A284M; ++ I287T; N288S; E296W; D324L; T332V; G353E;613/614 S29R; N94K; S137N; G157R; V184Q; Q220H; L223T; S232A; H259V;E261I; +++ A279V; A284M; I287T; N288S; E296W; D324L; T332V; G353E;615/616 S29R; N94R; G157R; V184Q; Q220H; L223T; S232A; H259V; E261I;A279F; +++ A284M; I287T; N288S; E296W; D324L; T332V; G353E; 617/618S29R; N94K; A111R; S137N; A154Y; G157R; V184Q; Q220H; L223T; S232A; +++H259V; E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 619/620S29R; N94R; G157R; V184Q; Q220H; L223T; S232A; H259V; E261I; A284M; +I287T; N288S; D324L; T332V; G353E; 621/622 S29R; N94R; G157R; V184Q;Q220H; L223T; S232A; H259V; E261I; A279V; +++ A284M; I287T; N288S;E296W; D324L; T332V; G353E; 623/624 S29R; G157R; V184Q; Q220H; L223T;S232A; H259V; E261I; A279V; A284M; ++ I287T; N288S; E296W; D324L; T332V;G353E; ¹Levels of increased activity were determined relative to theactivity of the reference polypeptide of SEQ ID NO: 448 and defined asfollows: “+” = at least 1.3-fold but less than 2-fold increasedactivity; “++” = at least 2-fold but less than 3-fold increasedactivity; “+++” = at least 3-fold increased activity but less than5-fold. ²Substrate compound (1j) activity assay: Lysate preparation, HTPassay reaction, and work-up and analysis were carried out as describedin Note 3, Table 3G.

TABLE 3J Engineered Polypeptides and Relative Enzyme Improvements UsingHTP Preparations SEQ Increased ID Activity¹ NO: Amino Acid Differences(compound (nt/aa) (Relative to SEQ ID NO: 96) (1j) assay) 625/626 S29R;N94R; A111R; S137N; G157R; G177H; V184Q; Q220H; L223T; S232A; + H259V;E261I; Q265L; A279V; A284M; I287T; N288S; A311T; D324L; T332V; G353E;627/628 S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A;H259V; ++ E261I; Q265L; S266T; A279V; A284M; I287T; N288S; F295S; A311V;D324L; S328A; T332V; G353E; 629/630 S29R; L71V; N94K; A111R; S137N;G157R; Y183C; V184Q; Q220H; L223T; ++ S232A; H259V; E261I; S266T; A279V;A284M; I287T; N288S; F295S; A311V; D324L; T332V; G353E; 631/632 S29R;N94K; A111R; S137N; G157R; G177H; V184Q; Q220H; L223T; S232A; + H259V;E261I; A279V; A284M; I287T; N288S; A311V; D324L; T332V; G353E; 633/634S29R; L71V; N94R; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; ++H259V; E261I; A279V; A284M; I287T; N288S; F295S; A311V; D324L; T332V;G353E; 635/636 S29R; N94T; A111R; S137N; G157R; G177H; V184Q; Q220H;L223T; S232A; ++ H259V; E261I; A279V; A284M; I287T; N288S; F295S; A311V;D324L; S328E; T332V; G353E; 637/638 S29R; N94R; A111R; S137N; G157R;Y183C; V184Q; Q220H; L223T; S232A; ++ H259V; E261I; A279V; A284M; I287T;N288S; A311T; D324L; S328E; T332V; G353E; 639/640 S29R; N94K; A111R;S137N; G157R; V184Q; Q220H; L223T; S232A; H259V; + E261I; S266T; A279V;A284M; I287T; N288S; F295S; A311V; D324L; T332V; G353E; 641/642 S29R;N94R; A111R; S137N; G157L; Y183C; V184Q; Q220H; L223T; S232A; + H259V;E261I; Q265L; A279V; A284M; I287T; N288S; A311V; D324L; T332V; G353E;643/644 S29R; L71C; N94R; A111R; S137N; G157L; G177H; V184Q; Q220H;L223T; +++ S232A; H259V; E261I; S266T; A279V; A284M; I287T; N288S;A311V; D324L; S328E; T332V; G353E; 645/646 S29R; N94K; A111R; S137N;G157R; G177H; Y183C; V184Q; Q220H; L223T; ++ S232A; H259V; E261I; Q265L;S266T; A279V; A284M; I287T; N288S; D324L; S328E; T332V; G353E; 647/648S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; H259V; ++E261I; S266T; A279V; A284M; I287T; N288S; F295S; A311V; D324L; S328E;T332V; G353E; 649/650 S29R; N94K; A111R; S137N; A154Y; G157L; V184Q;Q220H; L223T; S232A; ++ H259V; E261I; A279V; A284M; I287T; N288S; A311V;D324L; T332V; G353E; 651/652 S29R; L71V; N94K; A111R; S137N; G157L;V184Q; Q220H; L223T; S232A; ++ H259V; E261I; A279V; A284M; I287T; N288S;F295S; A311V; D324L; T332V; G353E; 653/654 S29R; L71C; N94R; A111R;S137N; G157R; G177H; V184Q; Q220H; L223T; + S232A; H259V; E261I; Q265L;A279V; A284M; I287T; N288S; F295S; A311V; D324L; S328A; T332V; G353E;655/656 S29R; N94K; A111R; S137N; G157L; G177H; V184Q; Q220H; L223T;S232A; ++ H259V; E261I; Q265L; A279V; A284M; I287T; N288S; A311V; D324L;S328E; T332V; G353E; 657/658 S29R; N94R; A111R; S137N; A154Y; G157R;V184Q; Q220H; L223T; S232A; ++ H259V; E261I; A279V; A284M; I287T; N288S;A311V; D324L; S328A; T332V; G353E; 659/660 S29R; N94K; A111R; S137N;A154Y; G157R; G177H; V184Q; Q220H; L223T; ++ S232A; H259V; E261I; Q265L;S266T; A279V; A284M; I287T; N288S; F295S; D324L; S328E; T332V; G353E;661/662 S29R; L71C; N94R; A111R; S137N; G157L; G177H; V184Q; Q220H;L223T; ++ S232A; H259V; E261I; A279V; A284M; I287T; N288S; A311V; D324L;T332V; G353E; 663/664 S29R; L71V; N94K; A111R; S137N; G157R; V184Q;Q220H; L223T; S232A; ++ H259V; E261I; Q265L; A279V; A284M; I287T; N288S;F295S; A311V; D324L; S328E; T332V; G353E; 665/666 S29R; N94K; A111R;S137N; G157L; V184Q; Q220H; L223T; S232A; H259V; +++ E261I; S266T;A279V; A284M; I287T; N288S; F295S; A311V; D324L; S328E; T332V; G353E;667/668 S29R; L71V; N94K; A111R; S137N; G157L; G177H; V184Q; Q220H;L223T; +++ S232A; H259V; E261I; S266T; A279V; A284M; I287T; N288S;A311V; D324L; S328E; T332V; G353E; 669/670 S29R; L71V; N94R; A111R;S137N; G157L; V184Q; Q220H; L223T; S232A; + H259V; E261I; Q265L; A279V;A284M; I287T; N288S; D324L; T332V; G353E; 671/672 S29R; N94K; A111R;S137N; G157L; G177H; V184Q; Q220H; L223T; S232A; ++ H259V; E261I; Q265L;A279V; A284M; I287T; N288S; A311V; D324L; T332V; G353E; 673/674 S29R;N94K; A111R; S137N; G157L; V184Q; Q220H; L223T; S232A; H259V; ++ E261I;A279V; A284M; I287T; N288S; A311T; D324L; S328E; T332V; G353E; 675/676S29R; L71V; N94K; A111R; S137N; G157R; Y183C; V184Q; Q220H; L223T; ++S232A; H259V; E261I; A279V; A284M; I287T; N288S; A311V; D324L; S328E;T332V; G353E; 677/678 S29R; L71C; N94K; A111R; S137N; G157L; G177H;V184Q; Q220H; L223T; ++ S232A; H259V; E261I; Q265L; A279V; A284M; I287T;N288S; A311V; D324L; T332V; G353E; 679/680 S29R; N94K; A111R; S137N;G157R; G177H; V184Q; Q220H; L223T; S232A; + H259V; E261I; A279V; A284M;I287T; N288S; A311T; D324L; S328E; T332V; G353E; 681/682 S29R; N94K;A111R; S137N; G157R; Y183C; V184Q; Q220H; L223T; S232A; ++ H259V; E261I;Q265L; S266T; A279V; A284M; I287T; N288S; A311V; D324L; T332V; G353E;683/684 S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A;H259V; + E261I; A279V; A284M; I287T; N288S; A311T; D324L; T332V; G353E;685/686 S29R; L71C; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T;S232A; + H259V; E261I; Q265L; A279V; A284M; I287T; N288S; A311V; D324L;T332V; G353E; 687/688 S29R; L71C; N94K; A111R; S137N; A154F; G157L;V184Q; Q220H; L223T; + S232A; H259V; E261I; Q265L; A279V; A284M; I287T;N288S; A311V; D324L; S328E; T332V; G353E; 689/690 S29R; N94K; A111R;S137N; G157L; G177H; V184Q; Q220H; L223T; S232A; + H259V; E261I; Q265L;A279V; A284M; I287T; N288S; A311T; D324L; T332V; G353E; 691/692 S29R;L71V; N94R; A111R; S137N; A154Y; G157R; V184Q; Q220H; L223T; ++ S232A;H259V; E261I; S266T; A279V; A284M; I287T; N288S; F295S; A311V; D324L;T332V; G353E; 693/694 S29R; N94K; A111R; S137N; G157R; G177H; V184Q;Q220H; L223T; S232A; +++ H259V; E261I; A279V; A284M; I287T; N288S;F295S; A311V; D324L; S328E; T332V; G353E; 695/696 S29R; N94R; A111R;S137N; G157L; V184Q; Q220H; L223T; S232A; H259V; ++ E261I; A279V; A284M;I287T; N288S; F295S; A311V; D324L; T332V; G353E; 697/698 S29R; L71C;N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; ++ H259V; E261I;A279V; A284M; I287T; N288S; F295S; A311V; D324L; T332V; G353E; 699/700S29R; N94K; A111R; S137N; G157R; G177H; Y183C; V184Q; Q220H; L223T; ++S232A; H259V; E261I; A279V; A284M; I287T; N288S; A311T; D324L; S328E;T332V; G353E; 701/702 S29R; L71V; N94R; A111R; S137N; G157L; G177H;V184Q; Q220H; L223T; + S232A; H259V; E261I; S266T; A279V; A284M; I287T;N288S; A311T; D324L; S328A; T332V; G353E; 703/704 S29R; L71C; N94K;A111R; S137N; G157R; Y183C; V184Q; Q220H; L223T; ++ S232A; H259V; E261I;S266T; A279V; A284M; I287T; N288S; F295S; A311V; D324L; T332V; G353E;705/706 S29R; L71V; N94R; A111R; S137N; G157L; V184Q; Q220H; L223T;S232A; +++ H259V; E261I; A279V; A284M; I287T; N288S; A311V; D324L;S328E; T332V; G353E; 707/708 S29R; N94K; A111R; S137N; G157L; G177H;V184Q; Q220H; L223T; S232A; + H259V; E261I; A279V; A284M; I287T; N288S;F295S; A311T; D324L; S328A; T332V; G353E; 709/710 S29R; L71V; N94K;A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; ++ H259V; E261I; Q265L;S266T; A279V; A284M; I287T; N288S; A311V; D324L; S328E; T332V; G353E;711/712 S29R; L71V; N94K; A111R; S137N; A154Y; G157R; G177H; V184Q;Q220H; ++ L223T; S232A; H259V; E261I; A279V; A284M; I287T; N288S; A311T;D324L; T332V; G353E; 713/714 S29R; L71V; N94T; A111R; S137N; G157R;G177H; V184Q; Q220H; L223T; ++ S232A; H259V; E261I; A279V; A284M; I287T;N288S; F295S; A311V; D324L; S328E; T332V; G353E; 715/716 S29R; N94R;A111R; S137N; G157L; Y183C; V184Q; Q220H; L223T; S232A; + H259V; E261I;Q265L; A279V; A284M; I287T; N288S; A311V; D324L; S328A; T332V; G353E;717/718 S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A;H259V; + E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 719/720S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; H259V; +E261I; N277I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 721/722S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; H259V; +E261I; A273W; A279V; A284M; I287T; N288S; D324L; I326V; T332V; G353E;723/724 S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A;H259V; + E261I; N277A; A279V; A284M; I287T; N288S; D324L; T332V; G353E;725/726 S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A;H259V; + E261I; A273W; A279V; A284M; I287T; N288S; D324L; T332V; G353E;727/728 S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A;H259V; + E261I; A279V; I283V; A284M; I287T; N288S; D324L; T332V; G353E;729/730 S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A;H259V; + E261I; A279L; A284M; I287T; N288S; D324L; T332V; G353E; 731/732S29R; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; H259W; +E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 733/734 S29R;N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; H259V; + E261I;V274M; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 735/736 S29R;N94K; A111R; S137N; A154F; G157R; V184Q; Q220H; L223T; S232A; + H259V;E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 737/738 S29R;N94K; A111R; S137N; N153V; G157R; V184Q; Q220H; L223T; S232A; ++ H259V;E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 739/740 S29R;N94K; A111R; S137N; N153Y; G157R; V184Q; Q220H; L223T; S232A; +++ H259V;E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 741/742 S29R;N94K; A111R; S137N; T141W; G157R; V184Q; Q220H; L223T; S232A; + H259V;E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 743/744 S29R;N94K; A111R; S137N; R143W; G157R; V184Q; Q220H; L223T; S232A; + H259V;E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 745/746 S29R;V82P; N94K; A111R; S137N; G157R; V184Q; Q220H; L223T; S232A; + H259V;E261I; A279V; A284M; I287T; N288S; D324L; T332V; G353E; 747/748 S29R;N94K; S137N; G157R; V184Q; Q220H; L223T; S232A; H259V; E261I; + A279V;A284M; I287T; N288S; E296V; D324L; T332V; G353E; 749/750 S29R; N94K;S137N; G157R; V184Q; Q220H; L223T; S232A; H259V; E261I; + A279V; A284M;I287T; N288S; E296N; D324L; T332V; G353E; ¹Levels of increased activitywere determined relative to the activity of the reference polypeptide ofSEQ ID NO: 598 and defined as follows: “+” = at least 1.3-fold but lessthan 2-fold increased activity; “++” = at least 2-fold but less than3-fold increased activity; “+++” = at least 3-fold increased activitybut less than 5-fold. ²Substrate compound (1j) activity assay: Lysatepreparation, HTP assay reaction, and work-up and analysis were carriedout as described in Note 3, Table 3G.

From an analysis of the exemplary polypeptides, improvements in enzymeproperties are associated with residue differences as compared to SEQ IDNO:2 at residue positions X4, X5, X14, X20, X29, X37, X67, X71, X74,X82, X94, X97, X100, X111, X124, X136, X137, X141, X143, X149, X153,X154, X156, X157, X158, X160, X163, X177, X178, X183, X184, X185, X186,X197, X198, X201, X220, X223, X226, X232, X243, X246, X256, X258, X259,X260, X261, X265, X266, X270, X273, X274, X277, X279, X280, X283, X284,X287, X288, X292, X293, X294, X295, X296, X297, X308, X311, X323, X324,X326, X328, X332, X353, and X356. The specific residue differences ateach of these positions that are associated with the improved propertiesinclude: X4H/L/R; X5T; X14P; X20T; X29R/T; X37H; X67A/D; X71C/V; X74R;X82P; X94K/R/T; X97P; X100W; X111M/Q/R/S; X124L/N; X136G; X137N; X141W;X143W; X149L; X153V/Y; X154F/M/Q/Y; X156G/I/Q/S/T/V; X157D/H/L/M/N/R;X158K; X160N; X163T; X177C/H; X178E; X183C; X184K/Q/R; X185V; X186K/R;X197I/P; X198A/E/H/P/S; X201L; X220D/H; X223T; X226L; X232A/R; X243G;X246W; X256V; X258D; X259E/H/I/L/M/S/T/V/W; X260G; X261A/G/I/K/R/S/T;X265G/L/Y; X266T; X270G; X273W; X274M; X277A/I; X279F/L/V/Y; X280L;X283V; X284K/L/M/Y; X287S/T; X288G/S; X292C/G/I/P/S/T/V/Y;X293H/I/K/L/N/Q/T/V; X294A/I/V; X295R/S; X296L/N/V/W; X297A; X308F;X311C/T/V; X323C/I/M/T/V; X324L/T; X326V; X328A/G/E; X332V; X353E; andX356R.

The specific enzyme properties associated with the residues differencesas compared to SEQ ID NO:4 at the residue positions above include, amongothers, enzyme activity, stereoselectivity, polypeptide expression.Improvements in enzyme activity are associated with residue differencesat residue positions X111, X136, X156, X197, X198, X201, X259, X280,X292, and X293. Improvements in selectivity for unactivated ketonesubstrates (as measured by increased activity in convertingcyclohexanone and L-norvaline to compound (3c)) are associated withresidue differences at residue positions: X197; X198; X201; X292; andincludes the specific amino acid residue differences: X197I/P;X198A/E/H/P/S; X201L; X292C/G/I/P/S/T/V/Y; and X293H/I/K/L/N/Q/T/V.Improvements in selectivity for unactivated amine substrates (asmeasured by increased activity in pyruvate and butylamine to compound(3b)) are associated with residue differences at residue positions:X111, X136, X156, X197P, X259, and X280; and includes the specific aminoacid residue differences: X111M/Q/S, X136G, X156G/I/Q/S/T/V, X197P,X259E/H/I/L/M/S/T, and X280L. Improvements in selectivity for thecombination of unactivated ketone and unactivated amine substrates (asmeasured by increased activity in converting cyclohexanone andbutylamine to compound (3d)) are associated with residue differences atresidue positions: X198, X259, and X280; and includes the specific aminoacid residue differences: X198E/H, X259M/H, and X280L. Accordingly, theresidue differences at the foregoing residue positions can be usedindividually or in various combinations to produce engineered iminereductase polypeptides having the desired improved properties,including, among others, enzyme activity, stereoselectivity, andsubstrate tolerance. Other residue differences affecting polypeptideexpression can be used to increase expression of the engineered iminereductase.

Improvements in enzyme activity and stability are associated withresidue differences at residue positions X4, X5, X14, X20, X29, X37,X67, X71, X74, X82, X94, X97, X100, X124, X137, X141, X143, X149, X153,X154, X157, X158, X160, X163, X177, X178, X183, X184, X185, X186, X220,X223, X226, X232, X243, X246, X256, X258, X260, X261, X265, X266, X270,X273, X274, X277, X279, X283, X284, X287, X288, X294, X295, X296, X297,X308, X311, X323, X324, X326, X328, X332, X353, and X356. Improvementsin activity and stability for the conversion of the ketone substratescompounds (1i) or compound (1j), and the amine substrate, butylamine(2b), to the amine products of compounds (3n) and (3o), respectively,are associated with the specific amino acid residue differences X4H/L/R,X5T, X14P, X20T, X29R/T, X37H, X67A/D, X71C/V, X74R, X82P, X94K/R/T,X97P, X100W, X111R, X124L/N, X137N, X141W, X143W, X149L, X153V/Y,X154F/M/Q/Y, X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H, X178E,X183C, X184K/Q/R, X185V, X186K/R, X220D/H, X223T, X226L, X232A/R, X243G,X246W, X256V, X258D, X259V/W, X260G, X261A/G/I/K/R/S/T, X265G/L/Y,X266T, X270G, X273W, X274M, X277A/I, X279F/L/V/Y, X283V, X284K/L/M/Y,X287S/T, X288G/S, X294A/I/V, X295R/S, X296L/N/V/W, X297A, X308F,X311C/T/V, X323C/I/M/T/V, X324L/T, X326V, X328A/G/E, X332V, X353E, andX356R.

Additionally, as noted in the Background, the crystal structure of theopine dehydrogenase CENDH has been determined (see Britton et al.,“Crystal structure and active site location ofN-(1-D-carboxyethyl)-L-norvaline dehydrogenase,” Nat. Struct. Biol.5(7): 593-601 (1998)). Accordingly, this correlation of the variousamino acid differences and functional activity disclosed herein alongwith the known three-dimensional structure of the wild-type enzyme CENDHcan provide the ordinary artisan with sufficient information torationally engineer further amino acid residue changes to thepolypeptides provided herein (and to homologous opine dehydrogenaseenzymes including OpDH, BADH, CEOS, and TauDH), and retain or improve onthe imine reductase activity properties. In some embodiments, it iscontemplated that such improvements can include engineering thenaturally occurring opine dehydrogenase polypeptides or the engineeredpolypeptides of the present disclosure to have imine reductase activitywith a range of substrates and provide a range of products as describedin Scheme 1.

In some embodiments, the present disclosure provides an engineeredpolypeptide having imine reductase activity, comprising an amino acidsequence having at least 80% sequence identity to a naturally occurringopine dehydrogenase amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 102, 104, 106, 108, and 110, and furthercomprising one or more residue differences as compared to the aminosequence of selected naturally occurring opine dehydrogenase. In someembodiments of the engineered polypeptide derived from an opinedehydrogenase, the imine reductase activity is the activity of Scheme 1,optionally, a reaction as disclosed in Table 2, and optionally, thereaction of converting compound (1b) and compound (2b) to productcompound (3d).

In light of the guidance provided herein, it is further contemplatedthat any of the exemplary engineered polypeptide sequences ofeven-numbered sequence identifiers SEQ ID NO: 4-100 and 112-750, orexemplary naturally occurring opine dehydrogenase polypeptides of SEQ IDNO: 2, 102, 104, 106, 108, and 110, can be used as the starting aminoacid sequence for synthesizing other engineered imine reductasepolypeptides, for example by subsequent rounds of evolution by addingnew combinations of various amino acid differences from otherpolypeptides in Tables 3A-3J, and other residue positions describedherein. Further improvements may be generated by including amino aciddifferences at residue positions that had been maintained as unchangedthroughout earlier rounds of evolution. Accordingly, in someembodiments, the engineered polypeptide having imine reductase activitycomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to reference sequence SEQ ID NO:2 and one or moreresidue differences as compared to SEQ ID NO:2 at residue positionsselected from: X111, X136, X156, X197, X198, X201, X259, X280, X292, andX293.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence having at least 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to reference sequence SEQ ID NO:2 and a residuedifference as compared to the sequence of SEQ ID NO: 2 at residueposition X198, wherein optionally the residue difference at positionX198 is selected from X198A, X198E, X198H, X198P, and X198S. In someembodiments, the engineered polypeptide having a residue difference atposition X198 comprises an amino acid sequence that is selected fromX198E, and X198H.

In some embodiments, the engineered polypeptide having imine reductaseactivity with improved properties as compared to SEQ ID NO:2, comprisesan amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to areference sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, and 100, and one or more residue differences as comparedto SEQ ID NO:2 at residue positions selected from: X111, X136, X156,X197, X198, X201, X259, X280, X292, and X293. In some embodiments, thereference sequence is selected from SEQ ID NO: 6, 50, 58, 60, 62, 64,72, 74, 76, 78, 88, 90, 92, 94, 96, 98 and 100. In some embodiments, thereference sequence is SEQ ID NO: 2. In some embodiments, the referencesequence is SEQ ID NO: 6. In some embodiments, the reference sequence isSEQ ID NO: 88. In some embodiments, the reference sequence is SEQ ID NO:90. In some embodiments, the reference sequence is SEQ ID NO: 92. Insome embodiments, the reference sequence is SEQ ID NO: 94. In someembodiments, the reference sequence is SEQ ID NO: 96. In someembodiments, the reference sequence is SEQ ID NO: 98. In someembodiments, the reference sequence is SEQ ID NO: 100.

In some embodiments, the residue differences at residue positions X111,X136, X156, X197, X198, X201, X259, X280, X292, and X293 are selectedfrom X111M, X111Q, X111S, X136G, X156G, X156I, X156Q, X156S, X156T,X156V, X197I, X197P, X198A, X198E, X198H, X198P, X198S, X201L, X259E,X259H, X259I, X259L, X259M, X259S, X259T, X280L, X292C, X292G, X292I,X292P, X292S, X292T, X292V, X292Y, X293H, X293I, X293K, X293L, X293N,X293Q, X293T, and X293V.

Accordingly, in some embodiments, the engineered imine reductasepolypeptides displaying one or more of the improved properties describedherein can comprise an amino acid sequence having the amino acidsequence identity to a reference sequence as described above, and one ormore residue differences as compared to SEQ ID NO:2 selected from:X111M, X111Q, X111S, X136G, X156G, X156I, X156Q, X156S, X156T, X156V,X197I, X197P, X198A, X198E, X198H, X198P, X198S, X201L, X259E, X259H,X259I, X259L, X259M, X259S, X259T, X280L, X292C, X292G, X292I, X292P,X292S, X292T, X292V, X292Y, X293H, X293I, X293K, X293L, X293N, X293Q,X293T, and X293V.

In some embodiments, the engineered imine reductase has an amino acidsequence comprising at least one or more residue differences as comparedto SEQ ID NO:2 selected from: X198E, X198H, X259M, X259H, and X280L.

In some embodiments, the engineered imine reductase polypeptidecomprises an amino acid sequence having at least a combination ofresidues differences as compared to SEQ ID NO:2 selected from: (a)X111M, X156T, X198H, X259M, X280L, X292V, and X293H; (b) X156T, X197P,X198H, X259H, X280L, X292P, and X293H; (c) X111M, X136G, X156S, X197I,X198H, X201L, X259H, X280L, X292V, and X293H; (d) X197I, X198E, X259M,and X280L; (e) X156T, X197I, X198E, X201L, X259H, X280L, X292V, andX293H; (f) X111M, X136G, X198H, X259M, X280L, X292S, and X293H; and (g)X156V, X197P, X198E, X201L, X259M, X280L, and X292T.

As will be appreciated by the skilled artisan, in some embodiments, oneor a combination of residue differences above that is selected can bekept constant in the engineered imine reductases as a core sequence, andadditional residue differences at other residue positions incorporatedinto the core sequence to generate additional engineered imine reductasepolypeptides with improved properties. Accordingly, it is to beunderstood for any engineered imine reductase containing one or a subsetof the residue differences above, the present disclosure contemplatesother engineered imine reductases that comprise the one or subset of theresidue differences, and additionally one or more residue differences atthe other residue positions disclosed herein. By way of example and notlimitation, an engineered imine reductase comprising a residuedifference at residue position X280, can further incorporate one or moreresidue differences at the other residue positions, e.g., X111, X136,X156, X197, X198, X201, X259, X292, and X293. Another example is anengineered imine reductase comprising a residue difference at residueposition X156, which can further comprise one or more residuedifferences at the other residue positions, e.g., X111, X136, X197,X198, X201, X259, X280, X292, and X293.

Indeed, the engineered imine reductase polypeptide of SEQ ID NO: 96which comprises the combination of residue differences as compared toSEQ ID NO:2: X156T, X197I, X198E, X201L, X259H, X280L, X292V, and X293H,was further evolved to generate additional engineered imine reductasepolypeptides with improved activity and stability. These furtherimproved engineered imine reductase polypeptides comprise one or moreresidue differences as compared to the sequence of SEQ ID NO: 2 atresidue positions selected from X4, X5, X14, X20, X29, X37, X67, X71,X74, X82, X94, X97, X100, X111, X124, X137, X141, X143, X149, X153,X154, X157, X158, X160, X163, X177, X178, X183, X184, X185, X186, X220,X223, X226, X232, X243, X246, X256, X258, X259, X260, X261, X265, X266,X270, X273, X274, X277, X279, X283, X284, X287, X288, X294, X295, X296,X297, X308, X311, X323, X324, X326, X328, X332, X353, and X356. Thespecific amino acid residue differences at these positions associatedwith improved activity or stability are selected from X4H/L/R, X5T,X14P, X20T, X29R/T, X37H, X67A/D, X71C/V, X74R, X82P, X94K/R/T, X97P,X100W, X111R, X124L/N, X137N, X141W, X143W, X149L, X153V/Y, X154F/M/Q/Y,X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H, X178E, X183C, X184K/Q/R,X185V, X186K/R, X220D/H, X223T, X226L, X232A/R, X243G, X246W, X256V,X258D, X259V/W, X260G, X261A/G/I/K/R/S/T, X265G/L/Y, X266T, X270G,X273W, X274M, X277A/I, X279F/L/V/Y, X283V, X284K/L/M/Y, X287S/T,X288G/S, X294A/I/V, X295R/S, X296L/N/V/W, X297A, X308F, X311C/T/V,X323C/I/M/T/V, X324L/T, X326V, X328A/G/E, X332V, X353E, and X356R.

Accordingly, in some embodiments the engineered polypeptide having iminereductase activity comprises an amino acid sequence having at least 80%sequence identity to SEQ ID NO: 2 (or any of the exemplary engineeredpolypeptides of SEQ ID NO: 4-100), one or more residue differences ascompared to the sequence of SEQ ID NO: 2 at residue positions selectedfrom X111, X136, X156, X197, X198, X201, X259, X280, X292, and X293 (asdescribed above), and further comprises one or more residue differencesas compared to the sequence of SEQ ID NO: 2 at residue positionsselected from X4, X5, X14, X20, X29, X37, X67, X71, X74, X82, X94, X97,X100, X111, X124, X137, X141, X143, X149, X153, X154, X157, X158, X160,X163, X177, X178, X183, X184, X185, X186, X220, X223, X226, X232, X243,X246, X256, X258, X259, X260, X261, X265, X266, X270, X273, X274, X277,X279, X283, X284, X287, X288, X294, X295, X296, X297, X308, X311, X323,X324, X326, X328, X332, X353, and X356. In some embodiments, thesefurther residue differences are selected from X4H/L/R, X5T, X14P, X20T,X29R/T, X37H, X67A/D, X71C/V, X74R, X82P, X94K/R/T, X97P, X100W, X111R,X124L/N, X137N, X141W, X143W, X149L, X153V/Y, X154F/M/Q/Y,X157D/H/L/M/N/R, X158K, X160N, X163T, X177C/H, X178E, X183C, X184K/Q/R,X185V, X186K/R, X220D/H, X223T, X226L, X232A/R, X243G, X246W, X256V,X258D, X259V/W, X260G, X261A/G/I/K/R/S/T, X265G/L/Y, X266T, X270G,X273W, X274M, X277A/I, X279F/L/V/Y, X283V, X284K/L/M/Y, X287S/T,X288G/S, X294A/I/V, X295R/S, X296L/N/V/W, X297A, X308F, X311C/T/V,X323C/I/M/T/V, X324L/T, X326V, X328A/G/E, X332V, X353E, X356R.

In some embodiments, the engineered polypeptide having imine reductaseactivity can comprise an amino acid sequence having at least 80%sequence identity to SEQ ID NO: 2, one or more residue differences ascompared to the sequence of SEQ ID NO: 2 at residue positions selectedfrom X111, X136, X156, X197, X198, X201, X259, X280, X292, and X293 (asdescribed above), and further comprise at least a combination of residuedifferences selected from: (a) X29R, X184R, X223T, X261S, X284M, andX287T; (b) X29R, X157R, X184Q, X220H, X223T, X232A, X261I, X284M, X287T,X288S, X324L, X332V, and X353E; (c) X29R, X157R, X184Q, X220H, X223T,X232A, X259V, X261I, X284M, X287T, X288S, X324L, X332V, and X353E; (d)X29R, X94K, X111R, X137N, X157R, X184Q, X220H, X223T, X232A, X259V,X261I, X279V, X284M, X287T, X288S, X324L, X332V, and X353E; and (e)X29R, X94K, X111R, X137N, X157R, X184Q, X220H, X223T, X232A, X259V,X261I, X266T, X279V, X284M, X287T, X288S, X295S, X311V, X324L, X328E,X332V, and X353E.

In some embodiments, the engineered polypeptide having imine reductaseactivity comprises an amino acid sequence having at least 80% sequenceidentity to SEQ ID NO: 2 and the combination of residue differencesX156T, X197I, X198E, X201L, X259H, X280L, X292V, and X293H, and furthercomprising one or more residue differences selected from X29R/T,X94K/R/T, X111R, X137N, X157D/H/L/M/N/R, X184K/Q/R, X220D/H, X223T,X232A/R, X259V/W, X261A/G/I/K/R/S/T, X266T, X279F/L/V/Y, X284K/L/M/Y,X287S/T, X288G/S, X295S, X311V, X324L/T, X328E, X332V, and X353E. Insome embodiment, the sequence comprises the combination of residuedifferences X156T, X197I, X198E, X201L, X259H, X280L, X292V, and X293H,and further comprises at least a combination of residue differencesselected from: (a) X29R, X184R, X223T, X261S, X284M, and X287T; (b)X29R, X157R, X184Q, X220H, X223T, X232A, X261I, X284M, X287T, X288S,X324L, X332V, and X353E; (c) X29R, X157R, X184Q, X220H, X223T, X232A,X259V, X261I, X284M, X287T, X288S, X324L, X332V, and X353E; (d) X29R,X94K, X111R, X137N, X157R, X184Q, X220H, X223T, X232A, X259V, X261I,X279V, X284M, X287T, X288S, X324L, X332V, and X353E; and (e) X29R, X94K,X111R, X137N, X157R, X184Q, X220H, X223T, X232A, X259V, X261I, X266T,X279V, X284M, X287T, X288S, X295S, X311V, X324L, X328E, X332V, andX353E.

Generally, the engineered polypeptides having imine reductase activityof the present disclosure are capable of converting a compound offormula (I) and an compound of formula (II) to an amine product compoundof formula (III) (as illustrated by Scheme 1) with improved activityand/or improved stereoselectivity relative to the Arthrobacter Sp.Strain C1 wild-type opine dehydrogenase reference polypeptide of SEQ IDNO: 2, or relative to a reference polypeptide having imine reductaseactivity selected from the engineered polypeptides of even-numberedsequence identifiers SEQ ID NO: 4-100 and 112-750. In some embodiments,the improved activity and/or improved stereoselectivity is with respectto the conversion of a specific combination of a compound of formula (I)and a compound of formula (II) shown in Table 2 to the correspondingamine product compound of formula (III) shown in Table 2.

Accordingly, in some embodiments, the engineered polypeptides havingimine reductase activity of the present disclosure which have an aminoacid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a referencesequence selected from even-numbered sequence identifiers SEQ ID NO:4-100 and 112-750, and one or more residue differences as compared toSEQ ID NO:2 at residue positions selected from: X4, X5, X14, X20, X29,X37, X67, X71, X74, X82, X94, X97, X100, X111, X124, X136, X137, X141,X143, X149, X153, X154, X156, X157, X158, X160, X163, X177, X178, X183,X184, X185, X186, X197, X198, X201, X220, X223, X226, X232, X243, X246,X256, X258, X259, X260, X261, X265, X266, X270, X273, X274, X277, X279,X280, X283, X284, X287, X288, X292, X293, X294, X295, X296, X297, X308,X311, X323, X324, X326, X328, X332, X353, and X356, are capable of oneor more of the following conversion reactions, under suitable reactionconditions, with improved activity and/or improved stereoselectivityrelative to a reference polypeptide of even-numbered sequenceidentifiers SEQ ID NO: 4-100 and 112-750:

(a) conversion of substrate compounds (1a) and (2a) to product compound(3a);

(b) conversion of substrate compounds (1a) and (2b) to product compound(3b);

(c) conversion of substrate compounds (1b) and (2a) to product compound(3c);

(d) conversion of substrate compounds (1b) and (2b) to product compound(3d);

(e) conversion of substrate compounds (1b) and (2c) to product compound(3e);

(f) conversion of substrate compounds (1b) and (2d) to product compound(3f);

(g) conversion of substrate compounds (1c) and (2a) to product compound(3g);

(h) conversion of substrate compounds (1d) and (2a) to product compound(3h);

(i) conversion of substrate compounds (1e) and (2b) to product compound(3i);

(j) conversion of substrate compounds (1f) and (2b) to product compound(3j);

(k) conversion of substrate compounds (1g) and (2e) to product compound(3k);

(l) conversion of substrate compounds (1b) and (2f) to product compound(3l);

(m) conversion of substrate compounds (1h) and (2a) to product compound(3m);(n) conversion of substrate compounds (1i) and (2b) to product compound(3n); and(o) conversion of substrate compounds (1j) and (2b) to product compound(3o).

In some embodiments, the engineered polypeptide having imine reductaseactivity and capable of catalyzing one or more of the above conversionreactions (a)-(o), under suitable reaction conditions, with improvedactivity and/or stereoselectivity comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to one of even-numbered sequenceidentifiers SEQ ID NO: 2-100 and 112-750, and the amino acid residuedifferences as compared to SEQ ID NO:2 present in any one ofeven-numbered sequence identifiers SEQ ID NO: 4-100 and 112-750, asprovided in Tables 3A-3J. In some embodiments, the engineeredpolypeptide having imine reductase activity and capable of catalyzingone or more of the above conversion reactions (a)-(o), under suitablereaction conditions, with improved activity and/or stereoselectivity hasan amino acid sequence comprising a sequence selected from theeven-numbered sequence identifiers SEQ ID NO: 4-100 and 112-750.

In some embodiments, the engineered imine reductase polypeptide iscapable of converting the ketone substrate of compound (1a) and theamine substrate of compound (2b) to the amine product compound (3b) withat least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold ormore activity relative to the activity of the reference polypeptide ofSEQ ID NO: 2. In some embodiments, the engineered imine reductasepolypeptide capable of converting the ketone substrate of compound (1a)and the amine substrate of compound (2b) to the amine product compound(3b) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold,10 fold, 20 fold 30 fold, 40 fold, 50 fold or more activity relative tothe activity of the reference polypeptide of SEQ ID NO:2 comprises anamino acid sequence having one or more residue differences selectedfrom: X111M/Q/S, X136G, X156G/I/Q/S/T/V, X259E/H/I/L/M/S/T, and X280L.In some embodiments, the engineered imine reductase polypeptide capableof converting the ketone substrate of compound (1a) and the aminesubstrate of compound (2b) to the amine product compound (3b) with atleast 1.2 fold the activity relative to SEQ ID NO:2 and comprises anamino acid sequence selected from: SEQ ID NO: 8, 12, 14, 18, 20, 22, 24,26, 28, 30, 32, 36, 38, 42, 44, 46, 50, 78, and 84.

In some embodiments, the engineered imine reductase polypeptide iscapable of converting the ketone substrate of compound (1b) and theamine substrate of compound (2a) to the amine product compound (3c) withat least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold ormore activity relative to the activity of the reference polypeptide ofSEQ ID NO: 2. In some embodiments, the engineered imine reductasepolypeptide capable of converting the ketone substrate of compound (1b)and the amine substrate of compound (2a) to the amine product compound(3c) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold,10 fold, 20 fold 30 fold, 40 fold, 50 fold or more activity relative tothe activity of the reference polypeptide of SEQ ID NO:2 comprises anamino acid sequence having one or more residue differences selectedfrom: X197I/P, X198A/E/H/P/S, X201L, X292C/G/I/P/S/T/V/Y, andX293H/I/K/L/N/Q/T/V. In some embodiments, the engineered imine reductasepolypeptide is capable of converting the ketone substrate of compound(1b) and an amine substrate of compound (2a) to a secondary amineproduct compound (3c) with at least 1.2 fold the activity relative toSEQ ID NO:2 and comprises an amino acid sequence selected from: SEQ IDNO: 4, 6, 16, 40, 48, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, and 86.

As noted above, the wild-type opine dehydrogenase from Arthrobacter Sp.Strain C1 (CENDH) of SEQ ID NO: 2 from which the engineered polypeptidesof the present disclosure were derived has no detectable activity inconverting a ketone substrate of compound (1b) and an amine substrate ofcompound (2b) to a secondary amine product compound (3d). In someembodiments, however, the engineered polypeptides having imine reductaseactivity are capable of converting a ketone substrate of compound (1b)and an amine substrate of compound (2b) to a secondary amine productcompound (3d). Further, in some embodiments, the engineered iminereductase polypeptides are capable of converting the ketone substrate ofcompound (1b) and the amine substrate of compound (2b) to the amineproduct compound (3d) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold,4 fold, 5 fold, 10 fold or more activity relative to the activity of areference polypeptide of SEQ ID NO: 90, 92, or 94, each of which is anengineered polypeptide having at least detectable activity in thisconversion. In some embodiments, the engineered imine reductasepolypeptide capable of converting the ketone substrate of compound (1b)and the amine substrate of compound (2b) to the amine product compound(3d) with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold,10 fold, 20 fold 30 fold, 40 fold, 50 fold or more activity relative tothe activity of the reference polypeptide of SEQ ID NO: 90, 92, or 94comprises an amino acid sequence having a combination of residuedifferences selected from: (a) X111M, X156T, X198H, X259M, X280L, X292V,and X293H; (b) X156T, X197P, X198H, X259H, X280L, X292P, and X293H; (c)X111M, X136G, X156S, X197I, X198H, X201L, X259H, X280L, X292V, andX293H; (d) X197I, X198E, X259M, and X280L; (e) X156T, X197I, X198E,X201L, X259H, X280L, X292V, and X293H; (f) X111M, X136G, X198H, X259M,X280L, X292S, and X293H; and (g) X156V, X197P, X198E, X201L, X259M,X280L, and X292T. In some embodiments, the engineered imine reductasepolypeptide is capable of converting the ketone substrate of compound(1b) and the amine substrate of compound (2b) to the amine productcompound (3d) comprises an amino acid sequence selected from: SEQ ID NO:88, 90, 92, 94, 96, 98, and 100.

In addition to the positions of residue differences specified above, anyof the engineered imine reductase polypeptides disclosed herein canfurther comprise other residue differences relative to SEQ ID NO:2 atother residue positions, i.e., residue positions other than X111, X136,X156, X197, X198, X201, X259, X280, X292, and X293. Residue differencesat these other residue positions can provide for additional variationsin the amino acid sequence without adversely affecting the ability ofthe polypeptide to catalyze one or more of the above conversionreactions (a)-(o) from Table 2. Accordingly, in some embodiments, inaddition to the amino acid residue differences present in any one of theengineered imine reductase polypeptides selected from SEQ ID NO: 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, and 100, the sequence canfurther comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, or 1-50 residue differences at other amino acid residue positionsas compared to the SEQ ID NO:2. In some embodiments, the number of aminoacid residue differences as compared to the reference sequence can be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 30, 35, 40, 45 or 50 residue positions. In someembodiments, the number of amino acid residue differences as compared tothe reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 residue positions. The residuedifference at these other positions can be conservative changes ornon-conservative changes. In some embodiments, the residue differencescan comprise conservative substitutions and non-conservativesubstitutions as compared to the naturally occurring imine reductasepolypeptide of SEQ ID NO: 2.

In some embodiments, the present disclosure also provides engineeredpolypeptides that comprise a fragment of any of the engineered iminereductase polypeptides described herein that retains the functionalactivity and/or improved property of that engineered imine reductase.Accordingly, in some embodiments, the present disclosure provides apolypeptide fragment capable of catalyzing one or more of the aboveconversion reactions (a)-(o) of Table 2, under suitable reactionconditions, wherein the fragment comprises at least about 80%, 90%, 95%,96%, 97%, 98%, or 99% of a full-length amino acid sequence of anengineered imine reductase polypeptide of the present disclosure, suchas an exemplary engineered imine reductase polypeptide selected fromeven-numbered sequence identifiers SEQ ID NO: 4-100 and 112-750.

In some embodiments, the engineered imine reductase polypeptide can havean amino acid sequence comprising a deletion of any one of theengineered imine reductase polypeptides described herein, such as theexemplary engineered polypeptides of even-numbered sequence identifiersSEQ ID NO: 4-100 and 112-750. Thus, for each and every embodiment of theengineered imine reductase polypeptides of the disclosure, the aminoacid sequence can comprise deletions of one or more amino acids, 2 ormore amino acids, 3 or more amino acids, 4 or more amino acids, 5 ormore amino acids, 6 or more amino acids, 8 or more amino acids, 10 ormore amino acids, 15 or more amino acids, or 20 or more amino acids, upto 10% of the total number of amino acids, up to 10% of the total numberof amino acids, up to 20% of the total number of amino acids, or up to30% of the total number of amino acids of the imine reductasepolypeptides, where the associated functional activity and/or improvedproperties of the engineered imine reductase described herein ismaintained. In some embodiments, the deletions can comprise 1-2, 1-3,1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24,1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues. In someembodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30,35, 40, 45, or 50 amino acid residues. In some embodiments, thedeletions can comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues.

In some embodiments, the engineered imine reductase polypeptide hereincan have an amino acid sequence comprising an insertion as compared toany one of the engineered imine reductase polypeptides described herein,such as the exemplary engineered polypeptides of even-numbered sequenceidentifiers SEQ ID NO: 4-100 and 112-750. Thus, for each and everyembodiment of the imine reductase polypeptides of the disclosure, theinsertions can comprise one or more amino acids, 2 or more amino acids,3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6or more amino acids, 8 or more amino acids, 10 or more amino acids, 15or more amino acids, 20 or more amino acids, 30 or more amino acids, 40or more amino acids, or 50 or more amino acids, where the associatedfunctional activity and/or improved properties of the engineered iminereductase described herein is maintained. The insertions can be to aminoor carboxy terminus, or internal portions of the imine reductasepolypeptide.

In some embodiments, the engineered imine reductase polypeptide hereincan have an amino acid sequence comprising a sequence selected fromeven-numbered sequence identifiers SEQ ID NO: 4-100 and 112-750, andoptionally one or several (e.g., up to 3, 4, 5, or up to 10) amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35,1-40, 1-45, or 1-50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the number of amino acid sequencehas optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residuedeletions, insertions and/or substitutions. In some embodiments, thesubstitutions can be conservative or non-conservative substitutions.

In the above embodiments, the suitable reaction conditions for theengineered polypeptides can be those described in Tables 3A-3J.Accordingly, in some embodiments, the suitable reaction conditions areHTP assay conditions, which can comprise: 20 mM loading each of ketonesubstrate and amine substrate compounds; 5 mM NADH; 20-100 μL clearlysate from E. coli expressing engineered polypeptide of interest; 100mM potassium phosphate buffer, pH 8.5 or pH 10; and a reactiontemperature at about 25° C. (room temperature) for a reaction time ofabout 12 h-24 h. In some embodiments, the suitable reaction conditionsare those described for shake flask powder (SFP) assays, which cancomprise: 50 mM loading each of ketone substrate and amine substratecompounds; 4.5 mM (3 g/L) NADH; 5-50 g/L SFP of engineered polypeptideof interest; 100 mM sodium formate; 1 g/L formate dehydrogenase(FDH-101; commercially available from Codexis, Inc. Redwood City,Calif., USA); 100 mM potassium phosphate buffer, pH 8.5 or pH 10; and areaction temperature at about 30° C. for a reaction time of about 12h-24 h. Guidance for use of these foregoing HTP and SFP reactionconditions and the imine reductase polypeptides are given in, amongothers, Tables 3A-3J, and the Examples.

In some embodiments, the polypeptides of the disclosure can be in theform of fusion polypeptides in which the engineered polypeptides arefused to other polypeptides, such as, by way of example and notlimitation, antibody tags (e.g., myc epitope), purification sequences(e.g., His tags for binding to metals), and cell localization signals(e.g., secretion signals). Thus, the engineered polypeptides describedherein can be used with or without fusions to other polypeptides.

It is to be understood that the polypeptides described herein are notrestricted to the genetically encoded amino acids. In addition to thegenetically encoded amino acids, the polypeptides described herein maybe comprised, either in whole or in part, of naturally-occurring and/orsynthetic non-encoded amino acids. Certain commonly encounterednon-encoded amino acids of which the polypeptides described herein maybe comprised include, but are not limited to: the D-stereomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (Melte); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine(Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

In some embodiments, the engineered polypeptides can be in variousforms, for example, such as an isolated preparation, as a substantiallypurified enzyme, whole cells transformed with gene(s) encoding theenzyme, and/or as cell extracts and/or lysates of such cells. Theenzymes can be lyophilized, spray-dried, precipitated or be in the formof a crude paste, as further discussed below.

In some embodiments, the engineered polypeptides can be provided on asolid support, such as a membrane, resin, solid carrier, or other solidphase material. A solid support can be composed of organic polymers suchas polystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled pore glass (CPG), reverse phase silica or metal, such as goldor platinum. The configuration of a solid support can be in the form ofbeads, spheres, particles, granules, a gel, a membrane or a surface.Surfaces can be planar, substantially planar, or non-planar. Solidsupports can be porous or non-porous, and can have swelling ornon-swelling characteristics. A solid support can be configured in theform of a well, depression, or other container, vessel, feature, orlocation.

In some embodiments, the engineered polypeptides having imine reductaseactivity of the present disclosure can be immobilized on a solid supportsuch that they retain their improved activity, stereoselectivity, and/orother improved properties relative to the reference polypeptide of SEQID NO: 2. In such embodiments, the immobilized polypeptides canfacilitate the biocatalytic conversion of the ketone and amine substratecompounds of formula (I) and formula (II) to the amine product compoundof formula (III), (e.g., as in conversion reactions (a)-(o) of Table 2),and after the reaction is complete are easily retained (e.g., byretaining beads on which polypeptide is immobilized) and then reused orrecycled in subsequent reactions. Such immobilized enzyme processesallow for further efficiency and cost reduction. Accordingly, it isfurther contemplated that any of the methods of using the iminereductase polypeptides of the present disclosure can be carried outusing the same imine reductase polypeptides bound or immobilized on asolid support.

Methods of enzyme immobilization are well-known in the art. Theengineered polypeptides can be bound non-covalently or covalently.Various methods for conjugation and immobilization of enzymes to solidsupports (e.g., resins, membranes, beads, glass, etc.) are well known inthe art and described in e.g., Yi et al., “Covalent immobilization ofω-transaminase from Vibrio fluvialis JS17 on chitosan beads,” ProcessBiochemistry 42(5): 895-898 (May 2007); Martin et al., “Characterizationof free and immobilized (S)-aminotransferase for acetophenoneproduction,” Applied Microbiology and Biotechnology 76(4): 843-851(September 2007); Koszelewski et al., “Immobilization of ω-transaminasesby encapsulation in a sol-gel/celite matrix,” Journal of MolecularCatalysis B: Enzymatic, 63: 39-44 (April 2010); Truppo et al.,“Development of an Improved Immobilized CAL-B for the EnzymaticResolution of a Key Intermediate to Odanacatib,” Organic ProcessResearch & Development, published online: dx.doi.org/10.1021/op200157c;Hermanson, G. T., Bioconjugate Techniques, Second Edition, AcademicPress (2008); Mateo et al., “Epoxy sepabeads: a novel epoxy support forstabilization of industrial enzymes via very intense multipoint covalentattachment,” Biotechnology Progress 18(3):629-34 (2002); andBioconjugation Protocols: Strategies and Methods, In Methods inMolecular Biology, C. M. Niemeyer ed., Humana Press (2004); thedisclosures of each which are incorporated by reference herein. Solidsupports useful for immobilizing the engineered imine reductases of thepresent disclosure include but are not limited to beads or resinscomprising polymethacrylate with epoxide functional groups,polymethacrylate with amino epoxide functional groups, styrene/DVBcopolymer or polymethacrylate with octadecyl functional groups.Exemplary solid supports useful for immobilizing the engineered iminereductase polypeptides of the present disclosure include, but are notlimited to, chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi),including the following different types of SEPABEAD: EC-EP, EC-HFA/S,EXA252, EXE119 and EXE120.

In some embodiments, the polypeptides described herein can be providedin the form of kits. The enzymes in the kits may be present individuallyor as a plurality of enzymes. The kits can further include reagents forcarrying out the enzymatic reactions, substrates for assessing theactivity of enzymes, as well as reagents for detecting the products. Thekits can also include reagent dispensers and instructions for use of thekits.

In some embodiments, the kits of the present disclosure include arrayscomprising a plurality of different imine reductase polypeptides atdifferent addressable position, wherein the different polypeptides aredifferent variants of a reference sequence each having at least onedifferent improved enzyme property. In some embodiments, a plurality ofpolypeptides immobilized on solid supports can be configured on an arrayat various locations, addressable for robotic delivery of reagents, orby detection methods and/or instruments. The array can be used to test avariety of substrate compounds for conversion by the polypeptides. Sucharrays comprising a plurality of engineered polypeptides and methods oftheir use are described in, e.g., WO2009/008908A2.

6.4 Polynucleotides Encoding Engineered Imine Reductases, ExpressionVectors and Host Cells

In another aspect, the present disclosure provides polynucleotidesencoding the engineered imine reductase polypeptides described herein.The polynucleotides may be operatively linked to one or moreheterologous regulatory sequences that control gene expression to createa recombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered imine reductase can be introduced into appropriate hostcells to express the corresponding imine reductase polypeptide.

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode the improved imine reductase enzymes. Thus, havingknowledge of a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present disclosurespecifically contemplates each and every possible variation ofpolynucleotides that could be made encoding the polypeptides describedherein by selecting combinations based on the possible codon choices,and all such variations are to be considered specifically disclosed forany polypeptide described herein, including the amino acid sequencespresented in Tables 3A-3J and disclosed in the sequence listingincorporated by reference herein as even-numbered sequence identifiersSEQ ID NO: 4-100 and 112-750.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. In some embodiments, all codons need not be replaced to optimizethe codon usage of the imine reductases since the natural sequence willcomprise preferred codons and because use of preferred codons may not berequired for all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the imine reductase enzymes may containpreferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90%of codon positions of the full length coding region.

In some embodiments, the polynucleotide comprises a codon optimizednucleotide sequence encoding the naturally occurring imine reductasepolypeptide of SEQ ID NO:2. In some embodiments, the polynucleotide hasa nucleic acid sequence comprising at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identity to the codon optimized nucleicacid sequence of SEQ ID NO: 1. The codon optimized sequence of SEQ IDNO:1 enhances expression of the encoded, naturally occurring iminereductase, providing preparations of enzyme capable of converting invitro over 80% of compound (2) to compound (1) under mini-DSP Assayconditions, and converting over 45% of compound (2) to compound (1)under DSP Assay conditions.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference sequence of SEQ ID NO:1, or a complement thereof, and encodes a polypeptide having iminereductase activity.

In some embodiments, as described above, the polynucleotide encodes anengineered polypeptide having imine reductase activity with improvedproperties as compared to SEQ ID NO: 2, where the polypeptide comprisesan amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to areference sequence selected from even-numbered sequence identifiers SEQID NO: 4-100 and 112-750, and one or more residue differences ascompared to SEQ ID NO:2 at residue positions selected from: X4, X5, X14,X20, X29, X37, X67, X71, X74, X82, X94, X97, X100, X111, X124, X136,X137, X141, X143, X149, X153, X154, X156, X157, X158, X160, X163, X177,X178, X183, X184, X185, X186, X197, X198, X201, X220, X223, X226, X232,X243, X246, X256, X258, X259, X260, X261, X265, X266, X270, X273, X274,X277, X279, X280, X283, X284, X287, X288, X292, X293, X294, X295, X296,X297, X308, X311, X323, X324, X326, X328, X332, X353, and X356. In someembodiments, the residue differences at these residue positions areselected from: X4H/L/R; X5T; X14P; X20T; X29R/T; X37H; X67A/D; X71C/V;X74R; X82P; X94K/R/T; X97P; X100W; X111M/Q/R/S; X124L/N; X136G; X137N;X141W; X143W; X149L; X153V/Y; X154F/M/Q/Y; X156G/I/Q/S/T/V;X157D/H/L/M/N/R; X158K; X160N; X163T; X177C/H; X178E; X183C; X184K/Q/R;X185V; X186K/R; X197I/P; X198A/E/H/P/S; X201L; X220D/H; X223T; X226L;X232A/R; X243G; X246W; X256V; X258D; X259E/H/I/L/M/S/T/V/W; X260G;X261A/G/I/K/R/S/T; X265G/L/Y; X266T; X270G; X273W; X274M; X277A/I;X279F/L/V/Y; X280L; X283V; X284K/L/M/Y; X287S/T; X288G/S;X292C/G/I/P/S/T/V/Y; X293H/I/K/L/N/Q/T/V; X294A/I/V; X295R/S;X296L/N/V/W; X297A; X308F; X311C/T/V; X323C/I/M/T/V; X324L/T; X326V;X328A/G/E; X332V; X353E; and X356R. In some embodiments, the referencesequence is selected from SEQ ID NO: 6, 50, 58, 60, 62, 64, 72, 74, 76,78, 88, 90, 92, 94, 96, 98 and 100. In some embodiments, the referencesequence is SEQ ID NO: 2. In some embodiments, the reference sequence isSEQ ID NO: 6. In some embodiments, the reference sequence is SEQ ID NO:88. In some embodiments, the reference sequence is SEQ ID NO: 90. Insome embodiments, the reference sequence is SEQ ID NO: 92. In someembodiments, the reference sequence is SEQ ID NO: 94. In someembodiments, the reference sequence is SEQ ID NO: 96. In someembodiments, the reference sequence is SEQ ID NO: 98. In someembodiments, the reference sequence is SEQ ID NO: 100.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1b) and (2b) tothe product compound (3d) with improved properties as compared to SEQ IDNO:2, wherein the polypeptide comprises an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to reference sequence SEQ IDNO:2 and one or more residue differences as compared to SEQ ID NO: 2 atresidue positions selected from: X111, X136, X156, X197, X198, X201,X259, X280, X292, and X293.

In some embodiments, the polynucleotide encodes a imine reductasepolypeptide capable of converting substrate compounds (1b) and (2b) tothe product compound (3d) with improved properties as compared to SEQ IDNO:2, wherein the polypeptide comprises an amino acid sequence having atleast 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to reference sequence SEQ IDNO:2, and at least a combination of residue differences as compared toSEQ ID NO: 2 selected from: (a) X111M, X156T, X198H, X259M, X280L,X292V, and X293H; (b) X156T, X197P, X198H, X259H, X280L, X292P, andX293H; (c) X111M, X136G, X156S, X197I, X198H, X201L, X259H, X280L,X292V, and X293H; (d) X197I, X198E, X259M, and X280L; (e) X156T, X197I,X198E, X201L, X259H, X280L, X292V, and X293H; (f) X111M, X136G, X198H,X259M, X280L, X292S, and X293H; and (g) X156V, X197P, X198E, X201L,X259M, X280L, and X292T.

In some embodiments, the polynucleotide encodes an engineered iminereductase polypeptide capable of converting substrate compounds (1b) and(2b) to the product compound (3d) with improved enzyme properties ascompared to the reference polypeptide of SEQ ID NO: 2, wherein thepolypeptide comprises an amino acid sequence having at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to a reference polypeptide selected from any one ofeven-numbered sequence identifiers SEQ ID NO: 4-100 and 112-750, withthe proviso that the amino acid sequence comprises any one of the set ofresidue differences as compared to SEQ ID NO: 2 contained in any one ofthe polypeptide sequences of even-numbered sequence identifiers SEQ IDNO: 4-100 and 112-750, as listed in Tables 3A-3J.

In some embodiments, the polynucleotide encoding the engineered iminereductase comprises an polynucleotide sequence selected from theodd-numbered sequence identifiers SEQ ID NO: 3-99 and 111-749.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from the odd-numbered sequence identifiers SEQ ID NO: 3-99 and111-749, or a complement thereof, and encodes a polypeptide having iminereductase activity with one or more of the improved properties describedherein. In some embodiments, the polynucleotide capable of hybridizingunder highly stringent conditions encodes a imine reductase polypeptidethat has an amino acid sequence that comprises one or more residuedifferences as compared to SEQ ID NO: 2 at residue positions selectedfrom: X4, X5, X14, X20, X29, X37, X67, X71, X74, X82, X94, X97, X100,X111, X124, X136, X137, X141, X143, X149, X153, X154, X156, X157, X158,X160, X163, X177, X178, X183, X184, X185, X186, X197, X198, X201, X220,X223, X226, X232, X243, X246, X256, X258, X259, X260, X261, X265, X266,X270, X273, X274, X277, X279, X280, X283, X284, X287, X288, X292, X293,X294, X295, X296, X297, X308, X311, X323, X324, X326, X328, X332, X353,and X356. In some embodiments, the specific residue differences at theseresidue positions are selected from: X4H/L/R; X5T; X14P; X20T; X29R/T;X37H; X67A/D; X71C/V; X74R; X82P; X94K/R/T; X97P; X100W; X111M/Q/R/S;X124L/N; X136G; X137N; X141W; X143W; X149L; X153V/Y; X154F/M/Q/Y;X156G/I/Q/S/T/V; X157D/H/L/M/N/R; X158K; X160N; X163T; X177C/H; X178E;X183C; X184K/Q/R; X185V; X186K/R; X197I/P; X198A/E/H/P/S; X201L;X220D/H; X223T; X226L; X232A/R; X243G; X246W; X256V; X258D;X259E/H/I/L/M/S/T/V/W; X260G; X261A/G/I/K/R/S/T; X265G/L/Y; X266T;X270G; X273W; X274M; X277A/I; X279F/L/V/Y; X280L; X283V; X284K/L/M/Y;X287S/T; X288G/S; X292C/G/I/P/S/T/V/Y; X293H/I/K/L/N/Q/T/V; X294A/I/V;X295R/S; X296L/N/V/W; X297A; X308F; X311C/T/V; X323C/I/M/T/V; X324L/T;X326V; X328A/G/E; X332V; X353E; and X356R.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity at the nucleotide level to areference polynucleotide encoding the engineered imine reductase. Insome embodiments, the reference polynucleotide sequence is selected fromthe odd-numbered sequence identifiers SEQ ID NO: 3-99 and 111-749.

An isolated polynucleotide encoding an improved imine reductasepolypeptide may be manipulated in a variety of ways to provide forexpression of the polypeptide. In some embodiments, the polynucleotidesencoding the polypeptides can be provided as expression vectors whereone or more control sequences is present to regulate the expression ofthe polynucleotides and/or polypeptides. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3^(rd)Ed., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

In some embodiments, the control sequences include among others,promoters, leader sequence, polyadenylation sequence, propeptidesequence, signal peptide sequence, and transcription terminator.Suitable promoters can be selected based on the host cells used. Forbacterial host cells, suitable promoters for directing transcription ofthe nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. NatlAcad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer etal., 1983, Proc. Natl Acad. Sci. USA 80: 21-25). Exemplary promoters forfilamentous fungal host cells, include promoters obtained from the genesfor Aspergillus oryzae TAKA amylase, Rhizomucor miehei asparticproteinase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-likeprotease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of thepromoters from the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase), and mutant, truncated,and hybrid promoters thereof. Exemplary yeast cell promoters can be fromthe genes can be from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomycescerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention. For example, exemplary transcription terminatorsfor filamentous fungal host cells can be obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. Any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention. Effectivesignal peptide coding regions for bacterial host cells are the signalpeptide coding regions obtained from the genes for Bacillus NCIB 11837maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacilluslicheniformis subtilisin, Bacillus licheniformis beta-lactamase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are described by Simonenand Palva, 1993, Microbiol Rev 57: 109-137. Effective signal peptidecoding regions for filamentous fungal host cells can be the signalpeptide coding regions obtained from the genes for Aspergillus oryzaeTAKA amylase, Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolenscellulase, and Humicola lanuginosa lipase. Useful signal peptides foryeast host cells can be from the genes for Saccharomyces cerevisiaealpha-factor and Saccharomyces cerevisiae invertase. Other useful signalpeptide coding regions are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila lactase (WO 95/33836). Whereboth signal peptide and propeptide regions are present at the aminoterminus of a polypeptide, the propeptide region is positioned next tothe amino terminus of a polypeptide and the signal peptide region ispositioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide of thepresent invention would be operably linked with the regulatory sequence.

In another aspect, the present disclosure is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered imine reductase polypeptide, and one or more expressionregulating regions such as a promoter and a terminator, a replicationorigin, etc., depending on the type of hosts into which they are to beintroduced. The various nucleic acid and control sequences describedabove may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding thepolypeptide at such sites. Alternatively, the nucleic acid sequence ofthe present disclosure may be expressed by inserting the nucleic acidsequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol (Example 1) or tetracycline resistance.Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3. Selectable markers for use in a filamentous fungal hostcell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Embodiments for use in an Aspergillus cell includethe amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzaeand the bar gene of Streptomyces hygroscopicus.

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved imine reductasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe imine reductase enzyme in the host cell. Host cells for use inexpressing the polypeptides encoded by the expression vectors of thepresent invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Bacillus subtilis,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. Exemplary host cells are Escherichiacoli W3110 (ΔfhuA) and BL21.

Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art. Polynucleotidesfor expression of the imine reductase may be introduced into cells byvarious methods known in the art. Techniques include among others,electroporation, biolistic particle bombardment, liposome mediatedtransfection, calcium chloride transfection, and protoplast fusion.

In some embodiments, the polypeptides can be expressed in cell freeexpression systems, for example those described in Kudlicki et al., CellFree Expression, 1^(st) Ed., Landes Biosciences (2007) and Cell FreeProtein Synthesis: Methods and Protocols, 1^(st) Ed., Spirin et al.,eds., Wiley-VCH (2007), all of which are incorporated herein byreference.

In the embodiments herein, the improved polypeptides and correspondingpolynucleotides can be obtained using methods used by those skilled inthe art. The engineered imine reductases described herein can beobtained by subjecting the polynucleotide encoding the naturallyoccurring gene encoding the wild-type opine dehydrogenase CENDH (SEQ IDNO: 2) or another engineered imine reductase to mutagenesis and/ordirected evolution methods, as discussed above. An exemplary directedevolution technique is mutagenesis and/or directed evolution methods(see, e.g., Stemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; PCTPubl. Nos. WO 95/22625, WO 97/0078, WO 97/35966, WO 98/27230, WO00/42651, and WO 01/75767; U.S. Pat. Nos. 6,537,746; 6,117,679;6,376,246; and 6,586,182; and U.S. Pat. Publ. Nos. 20080220990A1 and20090312196A1; each of which is hereby incorporated by referenceherein). Other directed evolution procedures that can be used include,among others, staggered extension process (StEP), in vitro recombination(Zhao et al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR(Caldwell et al., 1994, PCR Methods Appl. 3:S136-S140), and cassettemutagenesis (Black et al., 1996, Proc Natl Acad Sci USA 93:3525-3529).Mutagenesis and directed evolution techniques useful for the purposesherein are also described in the following references: Ling, et al.,1997, Anal. Biochem. 254(2):157-78; Dale et al., 1996,“Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod,” In Methods Mol. Biol. 57:369-74; Smith, 1985, Ann. Rev. Genet.19:423-462; Botstein et al., 1985, Science 229:1193-1201; Carter, 1986,Biochem. J. 237:1-7; Kramer et al., 1984, Cell, 38:879-887; Wells etal., 1985, Gene 34:315-323; Minshull et al., 1999, Curr Opin Chem Biol3:284-290; Christians et al., 1999, Nature Biotech 17:259-264; Crameriet al., 1998, Nature 391:288-291; Crameri et al., 1997, Nature Biotech15:436-438; Zhang et al., 1997, Proc Natl Acad Sci USA 94:45-4-4509;Crameri et al., 1996, Nature Biotech 14:315-319; Stemmer, 1994, Nature370:389-391; Stemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; WO95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767and U.S. Pat. No. 6,537,746. All publications are incorporated herein byreference.

The clones obtained following mutagenesis treatment can be screened forengineered imine reductases having one or more desired improved enzymeproperties. For example, where the improved enzyme property desired isincrease activity in the conversion of a ketone of compound (1b) and anamine of compound (2b) to a secondary amine of compound (3d), enzymeactivity may be measured for production of compound (3d). Clonescontaining a polynucleotide encoding a imine reductase with the desiredcharacteristics, e.g., increased production of compound (3d), are thenisolated, sequenced to identify the nucleotide sequence changes (ifany), and used to express the enzyme in a host cell. Measuring enzymeactivity from the expression libraries can be performed using thestandard biochemistry techniques, such as HPLC analysis and/orderivatization of products (pre or post separation), e.g., with dansylchloride or OPA.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides encodingportions of the imine reductase can be prepared by chemical synthesisusing, e.g., the classical phosphoramidite method described by Beaucageet al., 1981, Tet Lett 22:1859-69, or the method described by Matthes etal., 1984, EMBO J. 3:801-05, e.g., as it is typically practiced inautomated synthetic methods. According to the phosphoramidite method,oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer,purified, annealed, ligated and cloned in appropriate vectors. Inaddition, essentially any nucleic acid can be obtained from any of avariety of commercial sources. In some embodiments, additionalvariations can be created by synthesizing oligonucleotides containingdeletions, insertions, and/or substitutions, and combining theoligonucleotides in various permutations to create engineered iminereductases with improved properties.

Accordingly, in some embodiments, a method for preparing the engineeredimine reductases polypeptide can comprise: (a) synthesizing apolynucleotide encoding a polypeptide comprising an amino acid sequenceselected from the even-numbered sequence identifiers SEQ ID NO: 4-100and 112-750, and having one or more residue differences as compared toSEQ ID NO:2 at residue positions selected from: X4, X5, X14, X20, X29,X37, X67, X71, X74, X82, X94, X97, X100, X111, X124, X136, X137, X141,X143, X149, X153, X154, X156, X157, X158, X160, X163, X177, X178, X183,X184, X185, X186, X197, X198, X201, X220, X223, X226, X232, X243, X246,X256, X258, X259, X260, X261, X265, X266, X270, X273, X274, X277, X279,X280, X283, X284, X287, X288, X292, X293, X294, X295, X296, X297, X308,X311, X323, X324, X326, X328, X332, X353, and X356; and (b) expressingthe imine reductase polypeptide encoded by the polynucleotide.

In some embodiments of the method, the residue differences at residuepositions X4, X5, X14, X20, X29, X37, X67, X71, X74, X82, X94, X97,X100, X111, X124, X136, X137, X141, X143, X149, X153, X154, X156, X157,X158, X160, X163, X177, X178, X183, X184, X185, X186, X197, X198, X201,X220, X223, X226, X232, X243, X246, X256, X258, X259, X260, X261, X265,X266, X270, X273, X274, X277, X279, X280, X283, X284, X287, X288, X292,X293, X294, X295, X296, X297, X308, X311, X323, X324, X326, X328, X332,X353, and X356 are selected from X4H/L/R; X5T; X14P; X20T; X29R/T; X37H;X67A/D; X71C/V; X74R; X82P; X94K/R/T; X97P; X100W; X111M/Q/R/S; X124L/N;X136G; X137N; X141W; X143W; X149L; X153V/Y; X154F/M/I/Q/Y;X156G/I/Q/S/T/V; X157D/H/L/M/N/R; X158K; X160N; X163T; X177C/H; X178E;X183C; X184K/Q/R; X185V; X186K/R; X197I/P; X198A/E/H/P/S; X201L;X220D/H; X223T; X226L; X232A/R; X243G; X246W; X256V; X258D;X259E/H/I/L/M/S/T/V/W; X260G; X261A/G/I/K/R/S/T; X265G/L/Y; X266T;X270G; X273W; X274M; X277A/I; X279F/L/V/Y; X280L; X283V; X284K/L/M/Y;X287S/T; X288G/S; X292C/G/I/P/S/TN/Y; X293H/I/K/L/N/Q/T/V; X294A/I/V;X295R/S; X296L/N/V/W; X297A; X308F; X311C/T/V; X323C/I/M/T/V; X324L/T;X326V; X328A/G/E; X332V; X353E; and X356R.

In some embodiments of the method, the polynucleotide can encode anengineered imine reductase that has optionally one or several (e.g., upto 3, 4, 5, or up to 10) amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the number of amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

In another aspect, the present disclosure provides methods ofmanufacturing the engineered imine reductase polypeptides, where themethod can comprise culturing a host cell capable of expressing apolynucleotide encoding the imine reductase polypeptide under conditionssuitable for expression of the polypeptide. The method can furthercomprise isolating or purifying the expressed imine reductasepolypeptide, as described herein.

In some embodiments, the method for preparing or manufacturing theengineered imine reductase polypeptides further comprises the step ofisolating the polypeptide. The engineered polypeptides can be expressedin appropriate cells, as described above, and isolated (or recovered)from the host cells, the culture medium, and/or expression medium usingany one or more of the well known techniques used for proteinpurification, including, among others, lysozyme treatment, sonication,filtration, salting-out, ultra-centrifugation, and chromatography.Suitable solutions for lysing and the high efficiency extraction ofproteins from bacteria, such as E. coli, are commercially available,such as CelLytic B™ from Sigma-Aldrich of St. Louis Mo. Chromatographictechniques for isolation of the imine reductase polypeptides include,among others, reverse phase chromatography high performance liquidchromatography, ion exchange chromatography, gel electrophoresis, andaffinity chromatography.

In some embodiments, the non-naturally occurring polypeptides of thedisclosure can be prepared and used in various forms including but notlimited to crude extracts (e.g., cell-free lysates), powders (e.g.,shake-flask powders), lyophilizates, and substantially pure preparations(e.g., DSP powders), as further illustrated in the Examples below.

In some embodiments, the engineered polypeptides can be prepared andused in purified form, for example a substantially purified form.Generally, conditions for purifying a particular polypeptide willdepend, in part, on factors such as net charge, hydrophobicity,hydrophilicity, molecular weight, molecular shape, etc., and will beapparent to those having skill in the art. To facilitate purification,it is contemplated that in some embodiments the engineered polypeptidescan be expressed as fusion proteins with purification tags, such asHis-tags having affinity for metals, or antibody tags for binding toantibodies, e.g., myc epitope tag.

In some embodiments, affinity techniques may be used to isolate theimproved imine reductase enzymes. For affinity chromatographypurification, any antibody which specifically binds the imine reductasepolypeptide may be used. For the production of antibodies, various hostanimals, including but not limited to rabbits, mice, rats, etc., may beimmunized by injection with a imine reductase polypeptide, or a fragmentthereof. The imine reductase polypeptide or fragment may be attached toa suitable carrier, such as BSA, by means of a side chain functionalgroup or linkers attached to a side chain functional group. In someembodiments, the affinity purification can use a specific ligand boundby the imine reductase, such as poly(L-proline) or dye affinity column(see, e.g., EP0641862; Stellwagen, E., 2001, “Dye AffinityChromatography,” In Current Protocols in Protein Science Unit9.2-9.2.16).

6.5 Methods of Using the Engineered Imine Reductase Enzymes

In another aspect, the engineered polypeptides having imine reductaseactivity described herein can be used in a process for converting acompound of formula (I) and a compound of formula (II) to a secondary ortertiary amine compound of formula (III) as described above andillustrated in Scheme 1. Generally, such a biocatalytic process forcarrying out the reductive amination reaction of Scheme 1 comprisescontacting or incubating the ketone and amine substrate compounds withan engineered polypeptide having imine reductase activity of thedisclosure in the presence of a cofactor, such as NADH or NADPH, underreaction conditions suitable for formation of the amine product compoundof formula (III).

In some embodiments, the imine reductases can be used in a process forpreparing a secondary or tertiary amine product compound of formula(III),

wherein, R¹ and R² groups are independently selected from optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl; and optionally R¹ andR² are linked to form a 3-membered to 10-membered ring; R³ and R⁴ groupsare independently selected from a hydrogen atom, and optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl, with the proviso thatboth R³ and R⁴ cannot be hydrogen; and optionally R³ and R⁴ are linkedto form a 3-membered to 10-membered ring; and optionally, the carbonatom and/or the nitrogen indicated by * is chiral. The process comprisescontacting a ketone compound of formula (I),

wherein R¹, and R² are as defined above; and an amine compound offormula (II),

wherein R³, and R⁴ are as defined above; with an engineered polypeptidehaving imine reductase activity in presence of a cofactor under suitablereaction conditions.

As illustrated by the reactions in Table 2, and Tables 3A-3J, theengineered polypeptides having imine reductase activity of the presentdisclosure have activity with, or can be further engineered to haveactivity with, a wide range of amine substrate compounds of formula (II)in a process for preparing compound of formula (III). Accordingly, insome embodiments of the above biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), thecompound of formula (II) can be a primary amine wherein at least one ofR³ and R⁴ is hydrogen, whereby the product of formula (III) is asecondary amine compound. In some embodiments of the process, neither R³or R⁴ is hydrogen and the compound of formula (II) is a secondary amine,whereby the compound of formula (III) is tertiary amine. In someembodiments of the process, the compound of formula (II) is a secondaryamine and R³ or R⁴ are different, whereby the nitrogen atom indicatedby * of the amine compound of formula (III) is chiral. Further, in someembodiments, one stereoisomer of the chiral amine compound of formula(III) is formed stereoselectively, and optionally formed highlystereoselectively (e.g., in at least about 85% stereomeric excess).

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R³and R⁴ groups of the compound of formula (II) are linked to form a3-membered to 10-membered ring. In some embodiments, the ring is a5-membered to 8-membered is an optionally substituted cycloalkyl, aryl,arylalkyl, heterocycloalkyl, heteroaryl, or heteroarylalkyl ring.

In some embodiments of the biocatalytic process for preparing an amineproduct compound of formula (III), the compound of formula (II) is aprimary amine, wherein R³ group is hydrogen, and R⁴ is selected fromoptionally substituted (C₁-C₆)alkyl, (C₁-C₆)alkenyl, (C₁-C₆)alkynyl,(C₁-C₆)carboxyalkyl, (C₁-C₆)aminoalkyl, (C₁-C₆)haloalkyl, and(C₁-C₆)alkylthioalkyl. In some embodiments, the R⁴ group is selectedfrom optionally substituted (C₁-C₆)alkyl, (C₁-C₆)carboxyalkyl, and(C₁-C₆)aminoalkyl. In some embodiments, the R⁴ group is optionallysubstituted (C₁-C₆)alkyl, or (C₁-C₆)carboxyalkyl. In some embodiments,the compound of formula (II) is selected from methylamine,dimethylamine, isopropylamine, butylamine, and isobutylamine. In someembodiments, the amine substrate compound R³ group is hydrogen, and R⁴is selected from optionally substituted (C₄-C₈)cycloalkyl,(C₄-C₈)heterocycloalkyl, (C₄-C₈)aryl, (C₄-C₈)arylalkyl,(C₄-C₈)heteroaryl, and (C₄-C₈)heteroarylalkyl. In some embodiments, theamine substrate compound R³ group is hydrogen, and R⁴ is selected fromoptionally substituted (C₄-C₈)aryl, (C₄-C₈)arylalkyl, (C₄-C₈)heteroaryl,and (C₄-C₈)heteroarylalkyl. In some embodiments, the amine substratecompound R³ group is hydrogen, and R⁴ is optionally substituted(C₄-C₈)aryl. In some embodiments, the compound of formula (II) isoptionally substituted aniline.

As illustrated by the reactions in Table 2, and Tables 3A-3J, theengineered polypeptides having imine reductase activity of the presentdisclosure have activity with, or can be further engineered to haveactivity with, a wide range of ketone substrate compounds of formula (I)in a process for preparing compound of formula (III). In someembodiments, the R¹ and R² groups of the ketone substrate of compound(I) are different, whereby the carbon atom indicated by * of the aminecompound of formula (III) is chiral. Further, in some embodiments of theprocess, one stereoisomer of the chiral amine compound of formula (III)is formed stereoselectively, and optionally formed highlystereoselectively (e.g., in at least about 85% stereomeric excess).

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹and R² groups of the compound of formula (I) are linked to form a3-membered to 10-membered ring. In some embodiments, the ring is anoptionally substituted cycloalkyl, aryl, arylalkyl, heterocycloalkyl,heteroaryl, or heteroarylalkyl ring. In some embodiments of the process,the compound of formula (I) is selected from optionally substitutedcyclobutanone, cyclopentanone, cyclohexanone, and cycloheptanone.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹and R² groups of the compound of formula (I) are independently selectedfrom optionally substituted (C₁-C₆)alkyl, (C₁-C₆)alkenyl,(C₁-C₆)alkynyl, (C₁-C₆)carboxyalkyl, (C₁-C₆)aminoalkyl,(C₁-C₆)haloalkyl, and (C₁-C₆)alkylthioalkyl.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹group of the compound of formula (I) is selected from optionallysubstituted (C₁-C₆)alkyl, (C₁-C₆)alkenyl, (C₁-C₆)alkynyl,(C₁-C₆)carboxyalkyl, (C₁-C₆)aminoalkyl, (C₁-C₆)haloalkyl, and(C₁-C₆)alkylthioalkyl; and the R² group of the compound of formula (I)is selected from optionally substituted (C₄-C₈)cycloalkyl,(C₄-C₈)heterocycloalkyl, (C₄-C₈)arylalkyl, (C₄-C₈)heteroaryl, and(C₄-C₈)heteroarylalkyl.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹group of the compound of formula (I) is carboxy. In some embodiments,the compound of formula (I) is a 2-keto-acid selected from pyruvic acid,2-oxo-propanoic acid, 2-oxo-butanoic acid, 2-oxo-pentanoic acid,2-oxo-hexanoic acid.

In some embodiments of the biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), the R¹group of the compound of formula (I) is a hydrogen atom, and thecompound of formula (I) is an aldehyde. In such embodiments, the R¹group of the compound of formula (I) is selected from optionallysubstituted alkyl, alkenyl, alkynyl, alkoxy, carboxy, aminocarbonyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, aminoalkyl,haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl.

As illustrated by the compounds of formulas (I), (II), and (III), listedfor the reactions in Table 2, in some embodiments of the abovebiocatalytic process for preparing a secondary or tertiary amine productcompound of formula (III), the product compound of formula (III)comprises a compound selected from group consisting of: compound (3a),compound (3b), compound (3c), compound (3d), compound (3e), compound(3f), compound (3g), compound (3h), compound (3i), compound (3j),compound (3k), compound (3l), compound (3m), compound (3n), and compound(3o). In some embodiments of the process, the compound of formula (I)comprises a compound selected from group consisting of: compound (1a),compound (1b), compound (1e), compound (1d), compound (1e), compound(1f), compound (1g), compound (1h), compound (1i), and compound (1j). Insome embodiments of the process, the compound of formula (II) comprisesa compound selected from group consisting of: compound (2a), compound(2b), compound (2c), compound (2d), compound (2e), and compound (2f).

It is also contemplated that in some embodiments the process forpreparing an amine product compound of formula (III) catalyzed by anengineered polypeptide having imine reductase activity of the presentdisclosure comprises an intramolecular reaction, wherein the compound offormula (I) and the compound of formula (II) are groups on the same,single molecule. Thus, in some embodiments, at least one of R¹ and R² ofthe ketone compound of formula (I) is linked to at least one of R³ andR⁴ of the amine compound of formula (II), and the method comprisescontacting the single compound with a ketone group of formula (I) linkedto an amine group of formula (II) with an engineered polypeptide of thepresent disclosure under suitable reaction conditions. Illustrativeintramolecular reactions include but are not limited to reactions ofSchemes 2-5 shown below in Table 4, wherein groups R₁ and R₃ are asdefined above for groups R¹ and R³, and group R₅ is selected from ahydrogen atom, and optionally substituted alkyl, alkenyl, alkynyl,alkoxy, carboxy, aminocarbonyl, heteroalkyl, heteroalkenyl,heteroalkynyl, carboxyalkyl, aminoalkyl, haloalkyl, alkylthioalkyl,cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, andheteroarylalkyl.

TABLE 4 Scheme 2

Scheme 3

Scheme 4

Scheme 5

Without being bound by theory, it is believed that in most cases thebiocatalytic reaction of Scheme 1 involves the formation of anintermediate imine compound (e.g., an iminium intermediate) which isthen further reduced by the enzyme to the final secondary or tertiaryamine product compound of formula (III). It is also contemplated that insome embodiments, the process for preparing an amine product compound offormula (III) catalyzed by an engineered polypeptide having iminereductase activity of the present disclosure comprises contacting anengineered imine reductase polypeptide of the present disclosure with aketone compound of formula (I) and a primary amine compound of formula(II), whereby an imine intermediate is formed which then undergoes anintramolecular asymmetric cyclization reaction to yield a cyclicsecondary or tertiary hydroxyamine intermediate which undergoes hydroxylelimination to give a second imine (or enamine) intermediate. Thissecond imine (or enamine) is subsequently is then reduced in situ by theengineered imine reductase polypeptide of the present disclosure toyield the final cyclic amine product. Illustrative reactions involvingasymmetric cyclization through a hydroxyamine intermediate include butare not limited to reactions of Schemes 6-9 shown below in Table 5,wherein groups R₁ and R₃ are as defined above for groups R¹ and R³, andgroups R₅, R₆, and R₇ are independently selected from a hydrogen atom,and optionally substituted alkyl, alkenyl, alkynyl, alkoxy, carboxy,aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl,aminoalkyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl,heterocycloalkyl, heteroaryl, and heteroarylalkyl.

TABLE 5 Scheme 6

Scheme 7

Scheme 8

Scheme 9

Without being bound by theory, it is believed that the engineeredpolypeptides having imine reductase activity (IREDs) mediate not onlythe formation of the imine and/or hydroxyamine intermiates as shown inthe reactions of Schemes 2-9 but also the conversion of the imineintermediates to the final amine product compound of formula (III)depicted by the second reaction arrow.

Generally, imine compounds are less stable than amine compounds andsusceptible to undesirable oxidation reactions. It is contemplated,however, that in some embodiments of the processes of the presentdisclosure, an imine compound, or an enamine compound which cantautomerize to form an imine, can form from a ketone of formula (I) andan amine compound of formula (II) absent the presence of an enzyme, andthen be contacted with an engineered polypeptide of the presentdisclosure to catalyze its conversion of the final to a secondary ortertiary amine product compound of formula (III). For example, an imineor enamine intermediate compound first can be formed by combining aketone of formula (I) and an amine compound of formula (II) as shown inSchemes 6-9 but without the presence of an IRED (i.e., an engineeredpolypeptide having imine reductase activity). The imine compound formeddirectly or from tautomerization of an enamine compound can then becontacted with an engineered polypeptide having imine reductase activityto catalyze the conversion to the final amine product compound offormula (III). In some embodiments, it is contemplated that the imine orenamine intermediate compound, where suitably stable, can be isolatedbefore carrying out a step of contacting it with an engineeredpolypeptide having imine reductase activity. Thus, it is contemplatedthat in some embodiments of the process, an imine or enamine compound isformed first from the compounds of formula (I) and formula (II), orthrough the intramolecular reaction of compound having a ketone grouplinked to an amine group, and then this imine or enamine compound iscontacted with an engineered polypeptide having imine reductase activityto form an amine product compound of formula (III).

In some embodiments, a stable imine or enamine compound may be obtained(i.e., without first reacting a ketone compound of formula (I) and anamine compound of formula (II)) and used directly as a substrate with anIRED. It is contemplated that in such embodiments, the biocatalyticprocess is carried out wherein there is only a single substrate which isa stable imine or enamine compound, and this compound is contacted withan engineered polypeptide having imine reductase activity of the presentdisclosure which catalyzes the reduction of the stable imine compound toform a secondary compound of formula (III). In such a reaction, thestereoselectivity of the engineered polypeptide can mediate theformation of a chiral center adjacent to the amine group of the compoundof formula (III). Table 6 (below) lists three examples of stable iminecompounds that can undergo chiral reduction in a biocatalytic processwith an engineered polypeptide of the present disclosure to produceintermediate compounds for the synthesis of the pharmaceuticalssolifenacin and tadalafil, and for the synthesis of the pharmaceuticalcompound, dexmethylphenidate.

TABLE 6 Imine or Enamine Product Compound Substrate Compound of formula(III)

Alternatively, it is also contemplated that any of the product compoundsof formula (III) produced via an IRED catalyzed reaction of an isolatedimine or enamine substrate compound (as shown in Table 6) could also beproduced via an IRED-catalyzed intramolecular reaction (like thoseillustrated in Table 4) using as substrate the open-chain version of theimine or enamine substrate compounds. Accordingly, each of the productcompounds of Table 6 could also be made using the intermolecularsubstrate shown in Table 7 with an engineered polypeptide having iminereductase activity of the present disclosure.

TABLE 7 Intermolecular Substrate Compound (comprising formula (I)Product Compound and formula (II)) of formula (III)

There are numerous active pharmaceutical ingredient compounds thatinclude a secondary or tertiary amine group which could be produced viaa biocatalytic reductive amination using an engineered polypeptidehaving imine reductase activity of the present disclosure, and/or anengineered polypeptide produced by further directed evolution of anengineered polypeptide of the present disclosure. For example, Table 8lists various product compounds of formula (III) that are known activepharmaceutical ingredient compounds, or intermediate compounds usefulfor the synthesis of active pharmaceutical ingredient compounds, thatcould be produced using an engineered polypeptide having imine reductaseactivity of the present disclosure with the corresponding substratescompounds of formula (I) and/or formula (II).

TABLE 8 Substrate Compound Substrate Compound Product Compound offormula (I) of formula (II) of formula (III)

Me₂NH

Me₂NH

Me₂NH

In some embodiments of the above biocatalytic process for preparing asecondary or tertiary amine product compound of formula (III), theengineered polypeptide having imine reductase activity is derived from anaturally occurring opine dehydrogenase. In some embodiments, thenaturally occurring opine dehydrogenase is selected from: opinedehydrogenase from Arthrobacter sp. strain 1C (SEQ ID NO: 2), D-octopinedehydrogenase from Pecten maximus (SEQ ID NO: 102), ornithinedehydrogenase from Lactococcus lactis K1 (SEQ ID NO: 104),N-methyl-L-amino acid dehydrogenase from Pseudomonas putida (SEQ ID NO:106), β-alanopine dehydrogenase from Cellana grata (SEQ ID NO: 108), andtauropine dehydrogenase from Suberites domuncula (SEQ ID NO: 110). Insome embodiments, the engineered polypeptide having imine reductaseactivity is an engineered polypeptide derived from the opinedehydrogenase from Arthrobacter sp. strain 1C of SEQ ID NO: 2, asdisclosed herein, and exemplified by the engineered imine reductasepolypeptides of even numbered sequence identifiers SEQ ID NO: 4-100 and112-750.

Any of the engineered imine reductases described herein can be used inthe above biocatalytic processes for preparing a secondary or tertiaryamine compound of formula (III). By way of example and withoutlimitation, in some embodiments, the process can use an engineered iminereductase polypeptide comprising an amino acid sequence having at least80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more identity to a reference sequence selected fromeven-numbered sequence identifiers SEQ ID NO: 4-100 and 112-750. In someembodiments of the processes, the engineered imine reductase polypeptidecomprises an amino acid sequence having one or more residue differencesas compared to SEQ ID NO: 2 at residue positions X4, X5, X14, X20, X29,X37, X67, X71, X74, X82, X94, X97, X100, X111, X124, X136, X137, X141,X143, X149, X153, X154, X156, X157, X158, X160, X163, X177, X178, X183,X184, X185, X186, X197, X198, X201, X220, X223, X226, X232, X243, X246,X256, X258, X259, X260, X261, X265, X266, X270, X273, X274, X277, X279,X280, X283, X284, X287, X288, X292, X293, X294, X295, X296, X297, X308,X311, X323, X324, X326, X328, X332, X353, and X356. In some embodimentsof the processes, the residue differences at residue positions X4, X5,X14, X20, X29, X37, X67, X71, X74, X82, X94, X97, X100, X111, X124,X136, X137, X141, X143, X149, X153, X154, X156, X157, X158, X160, X163,X177, X178, X183, X184, X185, X186, X197, X198, X201, X220, X223, X226,X232, X243, X246, X256, X258, X259, X260, X261, X265, X266, X270, X273,X274, X277, X279, X280, X283, X284, X287, X288, X292, X293, X294, X295,X296, X297, X308, X311, X323, X324, X326, X328, X332, X353, and X356 areselected from X4H/L/R; X5T; X14P; X20T; X29R/T; X37H; X67A/D; X71C/V;X74R; X82P; X94K/R/T; X97P; X100W; X111M/Q/R/S; X124L/N; X136G; X137N;X141W; X143W; X149L; X153V/Y; X154F/M/Q/Y; X156G/I/Q/S/TN;X157D/H/L/M/N/R; X158K; X160N; X163T; X177C/H; X178E; X183C; X184K/Q/R;X185V; X186K/R; X197I/P; X198A/E/H/P/S; X201L; X220D/H; X223T; X226L;X232A/R; X243G; X246W; X256V; X258D; X259E/H/I/L/M/S/T/V/W; X260G;X261A/G/I/K/R/S/T; X265G/L/Y; X266T; X270G; X273W; X274M; X277A/I;X279F/L/V/Y; X280L; X283V; X284K/L/M/Y; X287S/T; X288G/S;X292C/G/I/P/S/TN/Y; X293H/M/L/N/Q/TN; X294A/I/V; X295R/S; X296L/N/V/W;X297A; X308F; X311C/T/V; X323C/I/M/T/V; X324L/T; X326V; X328A/G/E;X332V; X353E; and X356R.

In some embodiments of the above processes, the exemplary iminereductases capable of carrying out the conversion reactions (a)-(o) ofTable 2 disclosed herein, can be used. This includes the engineeredpolypeptides disclosed herein comprising an amino acid sequence selectedfrom even-numbered sequence identifiers SEQ ID NO: 4-100 and 112-750.Guidance on the choice and use of the engineered imine reductases isprovided in the descriptions herein, for example Tables 3A-3J, and theExamples.

In the embodiments herein and illustrated in the Examples, variousranges of suitable reaction conditions that can be used in theprocesses, include but are not limited to, substrate loading, cofactorloading, polypeptide loading, pH, temperature, buffer, solvent system,reaction time, and/or conditions with the polypeptide immobilized on asolid support. Further suitable reaction conditions for carrying out theprocess for biocatalytic conversion of substrate compounds to productcompounds using an engineered imine reductase polypeptide describedherein can be readily optimized in view of the guidance provided hereinby routine experimentation that includes, but is not limited to,contacting the engineered imine reductase polypeptide and ketone andamine substrate compounds of interest under experimental reactionconditions of concentration, pH, temperature, and solvent conditions,and detecting the product compound.

Generally, in the processes of the present disclosure, suitable reactionconditions include the presence of cofactor molecule which can act as anelectron donor in the reduction reaction carried out by the iminereductase. In some embodiments, the cofactor is selected from (but notlimited to) NADP⁺ (nicotinamide adenine dinucleotide phosphate), NADPH(the reduced form of NADP⁺), NAD⁺ (nicotinamide adenine dinucleotide)and NADH (the reduced form of NAD⁺). Generally, the reduced form of thecofactor is added to the enzyme reaction mixture. Accordingly, in someembodiments, the processes are carried out in presence of a cofactorselected from NADPH and NADH (these two cofactors are also referred toherein collectively as “NAD(P)H”). In some embodiments, the electrondonor is NADPH cofactor. In some embodiments, the process can be carriedout wherein the reaction conditions comprise an NADH or NADPH cofactorconcentration of about 0.03 to about 1 g/L, 0.03 to about 0.8 g/L, about0.03 to about 0.5 g/L, about 0.05 to about 0.3 g/L, about 0.05 to about0.2 g/L, or about 0.1 to about 0.2 g/L. In some embodiments, the processis carried out under NADH or NADPH cofactor concentration of about 1g/L, about 0.8 g/L, about 0.5 g/L, about 0.3 g/L, about 0.2 g/L, about0.1 g/L, about 0.05 g/L, or about 0.03 g/L.

In some embodiments of the process, an optional cofactor recyclingsystem, also referred to as a cofactor regeneration system, can be usedto regenerate cofactor NADPH/NADH from NADP+/NAD+ produced in theenzymatic reaction. A cofactor regeneration system refers to a set ofreactants that participate in a reaction that reduces the oxidized formof the cofactor (e.g., NADP⁺ to NADPH). Cofactors oxidized by thepolypeptide reduction of the keto substrate are regenerated in reducedform by the cofactor regeneration system. Cofactor regeneration systemscomprise a stoichiometric reductant that is a source of reducinghydrogen equivalents and is capable of reducing the oxidized form of thecofactor. The cofactor regeneration system may further comprise acatalyst, for example an enzyme catalyst, that catalyzes the reductionof the oxidized form of the cofactor by the reductant. Cofactorregeneration systems to regenerate NADH or NADPH from NAD⁺ or NADP⁺,respectively, are known in the art and can be used in the methodsdescribed herein.

Suitable exemplary cofactor regeneration systems that may be employed inthe imine reductase processes of the present disclosure include, but arenot limited to, formate and formate dehydrogenase, glucose and glucosedehydrogenase, glucose-6-phosphate and glucose-6-phosphatedehydrogenase, a secondary alcohol and alcohol dehydrogenase, phosphiteand phosphite dehydrogenase, molecular hydrogen and hydrogenase, and thelike. These systems may be used in combination with either NADP⁺/NADPHor NAD⁺/NADH as the cofactor. Electrochemical regeneration usinghydrogenase may also be used as a cofactor regeneration system. See,e.g., U.S. Pat. Nos. 5,538,867 and 6,495,023, both of which areincorporated herein by reference. Chemical cofactor regeneration systemscomprising a metal catalyst and a reducing agent (for example, molecularhydrogen or formate) may also be suitable. See, e.g., PCT publication WO2000/053731, which is incorporated herein by reference.

In some embodiments, the co-factor regenerating system comprises aformate dehydrogenase, which is a NAD⁺ or NADP⁺-dependent enzyme thatcatalyzes the conversion of formate and NAD⁺ or NADP⁺ to carbon dioxideand NADH or NADPH, respectively. Formate dehydrogenases suitable for useas cofactor regenerating systems in the imine reductase processesdescribed herein include naturally occurring and non-naturally occurringformate dehydrogenases. Suitable formate dehydrogenases are described inPCT publication WO 2005/018579, incorporated herein by reference. In oneembodiment, the formate dehydrogenase used in the process is FDH-101,which commercially available (Codexis, Inc. Redwood City, Calif., USA).Formate may be provided in the form of a salt, typically an alkali orammonium salt (for example, HCO₂Na, KHCO₂NH₄, and the like), in the formof formic acid, typically aqueous formic acid, or mixtures thereof. Abase or buffer may be used to provide the desired pH.

In some embodiments, the cofactor recycling system comprises glucosedehydrogenase (GDH), which is a NAD or NADP⁺-dependent enzyme thatcatalyzes the conversion of D-glucose and NAD⁺ or NADP⁺ to gluconic acidand NADH or NADPH, respectively. Glucose dehydrogenases suitable for usein the practice of the imine reductase processes described hereininclude naturally occurring glucose dehydrogenases as well asnon-naturally occurring glucose dehydrogenases. Naturally occurringglucose dehydrogenase encoding genes have been reported in theliterature, e.g., the Bacillus subtilis 61297 GDH gene, B. cereus ATCC14579 and B. megaterium. Non-naturally occurring glucose dehydrogenasesgenerated using, for example, mutagenesis, directed evolution, and thelike and are provided in PCT publication WO 2005/018579, and USpublication Nos. 2005/0095619 and 2005/0153417. All of these sequencesare incorporated herein by reference. In one embodiment, the glucosedehydrogenase used in the process is CDX-901 or GDH-105, each of whichcommercially available (Codexis, Inc. Redwood City, Calif., USA).

In some embodiments, the co-factor regenerating system comprises analcohol dehydrogenase or ketoreductase, which is an NAD⁺ orNADP⁺-dependent enzyme that catalyzes the conversion of a secondaryalcohol and NAD⁺ or NADP⁺ to a ketone and NADH or NADPH, respectively.Suitable secondary alcohols useful in cofactor regenerating systemsinclude lower secondary alkanols and aryl-alkyl carbinols, including butnot limited to, isopropanol, 2-butanol, 3-methyl-2-butanol, 2-pentanol,3-pentanol, 3,3-dimethyl-2-butanol, and the like. Alcohol dehydrogenasessuitable for use as cofactor regenerating systems in the processesdescribed herein include naturally occurring and non-naturally occurringketoreductases. Naturally occurring alcohol dehydrogenase/ketoreductaseinclude known enzymes from, by way of example and not limitation,Thermoanerobium brockii, Rhodococcus erythropolis, Lactobacillus kefir,and Lactobacillus brevis, and non-naturally occurring alcoholdehydrogenases include engineered alcohol dehydrogenases derivedtherefrom. In some embodiments, non-naturally occurring ketoreductasesengineered for thermo- and solvent stability can be used. Suchketoreductases are described in the present application and the patentpublications US 20080318295A1; US 20090093031A1; US 20090155863A1; US20090162909A1; US 20090191605A1; US 20100055751A1; WO/2010/025238A2;WO/2010/025287A2; and US 20100062499A1; each of which are incorporatedby reference herein.

The concentration of the ketone and amine substrate compounds in thereaction mixtures can be varied, taking into consideration, for example,the desired amount of product compound, the effect of substrateconcentration on enzyme activity, stability of enzyme under reactionconditions, and the percent conversion of the substrates to the product.In some embodiments, the suitable reaction conditions comprise asubstrate compound loading of at least about 0.5 to about 200 g/L, 1 toabout 200 g/L, 5 to about 150 g/L, about 10 to about 100 g/L, 20 toabout 100 g/L or about 50 to about 100 g/L. In some embodiments, thesuitable reaction conditions comprise loading of each of the ketone andamine substrate compounds of at least about 0.5 g/L, at least about 1g/L, at least about 5 g/L, at least about 10 g/L, at least about 15 g/L,at least about 20 g/L, at least about 30 g/L, at least about 50 g/L, atleast about 75 g/L, at least about 100 g/L, at least about 150 g/L or atleast about 200 g/L, or even greater. The values for substrate loadingsprovided herein are based on the molecular weight of compound (1b),however it also contemplated that the equivalent molar amounts of otherketone and amine substrates, such as ketone substrate compounds(1a)-(1j), and amine substrate compounds (2a)-(2f), could be used, aswell as equimolar amounts of hydrates or salts of any of these compoundscan be used in the process. It is also contemplated that in someembodiments, the suitable reaction conditions comprise loading of eachof the ketone and amine substrate compounds in terms of molarconcentrations equivalent to the above g/L concentrations for compound(1b). Thus, reaction conditions can comprise a substrate loading of eachof the ketone and amine substrate compounds of at least about 5 mM, atleast about 10 mM, at least about 25 mM, at least about 50 mM, at leastabout 75 mM, at least about 100 mM, or even greater. In addition, it iscontemplated that substrate compounds covered by formulas (I) and (II),can be used in the same ranges of amounts as those used for compound(1b).

In carrying out the imine reductase mediated processes described herein,the engineered polypeptide may be added to the reaction mixture in theform of a purified enzyme, partially purified enzyme, whole cellstransformed with gene(s) encoding the enzyme, as cell extracts and/orlysates of such cells, and/or as an enzyme immobilized on a solidsupport. Whole cells transformed with gene(s) encoding the engineeredimine reductase enzyme or cell extracts, lysates thereof, and isolatedenzymes may be employed in a variety of different forms, including solid(e.g., lyophilized, spray-dried, and the like) or semisolid (e.g., acrude paste). The cell extracts or cell lysates may be partiallypurified by precipitation (ammonium sulfate, polyethyleneimine, heattreatment or the like, followed by a desalting procedure prior tolyophilization (e.g., ultrafiltration, dialysis, and the like). Any ofthe enzyme preparations (including whole cell preparations) may bestabilized by cross-linking using known cross-linking agents, such as,for example, glutaraldehyde or immobilization to a solid phase (e.g.,Eupergit C, and the like).

The gene(s) encoding the engineered imine reductase polypeptides can betransformed into host cell separately or together into the same hostcell. For example, in some embodiments one set of host cells can betransformed with gene(s) encoding one engineered imine reductasepolypeptide and another set can be transformed with gene(s) encodinganother engineered imine reductase polypeptide. Both sets of transformedcells can be utilized together in the reaction mixture in the form ofwhole cells, or in the form of lysates or extracts derived therefrom. Inother embodiments, a host cell can be transformed with gene(s) encodingmultiple engineered imine reductase polypeptide. In some embodiments theengineered polypeptides can be expressed in the form of secretedpolypeptides and the culture medium containing the secreted polypeptidescan be used for the imine reductase reaction.

The improved activity and/or stereoselectivity of the engineered iminereductase polypeptides disclosed herein provides for processes whereinhigher percentage conversion can be achieved with lower concentrationsof the engineered polypeptide. In some embodiments of the process, thesuitable reaction conditions comprise an engineered polypeptide amountof about 1% (w/w), 2% (w/w), 5% (w/w), 10% (w/w), 20% (w/w), 30% (w/w),40% (w/w), 50% (w/w), 75% (w/w), 100% (w/w) or more of substratecompound loading.

In some embodiments, the engineered polypeptides are present at about0.01 g/L to about 50 g/L; about 0.05 g/L to about 50 g/L; about 0.1 g/Lto about 40 g/L; about 1 g/L to about 40 g/L; about 2 g/L to about 40g/L; about 5 g/L to about 40 g/L; about 5 g/L to about 30 g/L; about 0.1g/L to about 10 g/L; about 0.5 g/L to about 10 g/L; about 1 g/L to about10 g/L; about 0.1 g/L to about 5 g/L; about 0.5 g/L to about 5 g/L; orabout 0.1 g/L to about 2 g/L. In some embodiments, the imine reductasepolypeptide is present at about 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L,0.5 g/L, 1, 2 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35g/L, 40 g/L, or 50 g/L.

During the course of the reactions, the pH of the reaction mixture maychange. The pH of the reaction mixture may be maintained at a desired pHor within a desired pH range. This may be done by the addition of anacid or a base, before and/or during the course of the reaction.Alternatively, the pH may be controlled by using a buffer. Accordingly,in some embodiments, the reaction condition comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,by way of example and not limitation, borate, phosphate,2-(N-morpholino)ethanesulfonic acid (MES),3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine,and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), and the like. Insome embodiments, the buffer is phosphate. In some embodiments of theprocess, the suitable reaction conditions comprise a buffer (e.g.,phosphate) concentration is from about 0.01 to about 0.4 M, 0.05 toabout 0.4 M, 0.1 to about 0.3 M, or about 0.1 to about 0.2 M. In someembodiments, the reaction condition comprises a buffer (e.g., phosphate)concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12,0.14, 0.16, 0.18, 0.2, 0.3, or 0.4 M. In some embodiments, the reactionconditions comprise water as a suitable solvent with no buffer present.

In the embodiments of the process, the reaction conditions can comprisea suitable pH. The desired pH or desired pH range can be maintained byuse of an acid or base, an appropriate buffer, or a combination ofbuffering and acid or base addition. The pH of the reaction mixture canbe controlled before and/or during the course of the reaction. In someembodiments, the suitable reaction conditions comprise a solution pHfrom about 4 to about 10, pH from about 5 to about 10, pH from about 7to about 11, pH from about 8 to about 10, pH from about 6 to about 8. Insome embodiments, the reaction conditions comprise a solution pH ofabout 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

In the embodiments of the processes herein, a suitable temperature canbe used for the reaction conditions, for example, taking intoconsideration the increase in reaction rate at higher temperatures, andthe activity of the enzyme during the reaction time period. Accordingly,in some embodiments, the suitable reaction conditions comprise atemperature of from about 10° C. to about 80° C., about 10° C. to about70° C., about 15° C. to about 65° C., about 20° C. to about 60° C.,about 20° C. to about 55° C., about 25° C. to about 55° C., or about 30°C. to about 50° C. In some embodiments, the suitable reaction conditionscomprise a temperature of about 10° C., 15° C., 20° C., 25° C., 30° C.,35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C.,or 80° C. In some embodiments, the temperature during the enzymaticreaction can be maintained at a specific temperature throughout thecourse of the reaction. In some embodiments, the temperature during theenzymatic reaction can be adjusted over a temperature profile during thecourse of the reaction.

The processes of the disclosure are generally carried out in a solvent.Suitable solvents include water, aqueous buffer solutions, organicsolvents, polymeric solvents, and/or co-solvent systems, which generallycomprise aqueous solvents, organic solvents and/or polymeric solvents.The aqueous solvent (water or aqueous co-solvent system) may bepH-buffered or unbuffered. In some embodiments, the processes using theengineered imine reductase polypeptides can be carried out in an aqueousco-solvent system comprising an organic solvent (e.g., ethanol,isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF),dimethylacetamide (DMAc), N-methyl-pyrrolidone (NMP), ethyl acetate,butyl acetate, 1-octanol, heptane, octane, methyl t butyl ether (MTBE),toluene, and the like), ionic or polar solvents (e.g.,1-ethyl-4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, glycerol, polyethyleneglycol (PEG), and the like). In some embodiments, the co-solvent can bea polar solvent, such as a polyol, dimethylsulfoxide (DMSO), or loweralcohol. The non-aqueous co-solvent component of an aqueous co-solventsystem may be miscible with the aqueous component, providing a singleliquid phase, or may be partly miscible or immiscible with the aqueouscomponent, providing two liquid phases. Exemplary aqueous co-solventsystems can comprise water and one or more co-solvents selected from anorganic solvent, polar solvent, and polyol solvent. In general, theco-solvent component of an aqueous co-solvent system is chosen such thatit does not adversely inactivate the imine reductase enzyme under thereaction conditions. Appropriate co-solvent systems can be readilyidentified by measuring the enzymatic activity of the specifiedengineered imine reductase enzyme with a defined substrate of interestin the candidate solvent system, utilizing an enzyme activity assay,such as those described herein.

In some embodiments of the process, the suitable reaction conditionscomprise an aqueous co-solvent, where the co-solvent comprises DMSO atabout 1% to about 50% (v/v), about 1 to about 40% (v/v), about 2% toabout 40% (v/v), about 5% to about 30% (v/v), about 10% to about 30%(v/v), or about 10% to about 20% (v/v). In some embodiments of theprocess, the suitable reaction conditions can comprise an aqueousco-solvent comprising DMSO at about 1% (v/v), about 5% (v/v), about 10%(v/v), about 15% (v/v), about 20% (v/v), about 25% (v/v), about 30%(v/v), about 35% (v/v), about 40% (v/v), about 45% (v/v), or about 50%(v/v).

In some embodiments, the reaction conditions can comprise a surfactantfor stabilizing or enhancing the reaction. Surfactants can comprisenon-ionic, cationic, anionic and/or amphiphilic surfactants. Exemplarysurfactants, include by way of example and not limitations, nonylphenoxypolyethoxylethanol (NP40), Triton X-100,polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodiumoleylamidosulfate, polyoxyethylene-sorbitanmonostearate,hexadecyldimethylamine, etc. Any surfactant that may stabilize orenhance the reaction may be employed. The concentration of thesurfactant to be employed in the reaction may be generally from 0.1 to50 mg/ml, particularly from 1 to 20 mg/ml.

In some embodiments, the reaction conditions can include an antifoamagent, which aid in reducing or preventing formation of foam in thereaction solution, such as when the reaction solutions are mixed orsparged. Anti-foam agents include non-polar oils (e.g., minerals,silicones, etc.), polar oils (e.g., fatty acids, alkyl amines, alkylamides, alkyl sulfates, etc.), and hydrophobic (e.g., treated silica,polypropylene, etc.), some of which also function as surfactants.Exemplary anti-foam agents include, Y-30® (Dow Corning), poly-glycolcopolymers, oxy/ethoxylated alcohols, and polydimethylsiloxanes. In someembodiments, the anti-foam can be present at about 0.001% (v/v) to about5% (v/v), about 0.01% (v/v) to about 5% (v/v), about 0.1% (v/v) to about5% (v/v), or about 0.1% (v/v) to about 2% (v/v). In some embodiments,the anti-foam agent can be present at about 0.001% (v/v), about 0.01%(v/v), about 0.1% (v/v), about 0.5% (v/v), about 1% (v/v), about 2%(v/v), about 3% (v/v), about 4% (v/v), or about 5% (v/v) or more asdesirable to promote the reaction.

The quantities of reactants used in the imine reductase reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of imine reductase substrate employed. Thosehaving ordinary skill in the art will readily understand how to varythese quantities to tailor them to the desired level of productivity andscale of production.

In some embodiments, the order of addition of reactants is not critical.The reactants may be added together at the same time to a solvent (e.g.,monophasic solvent, biphasic aqueous co-solvent system, and the like),or alternatively, some of the reactants may be added separately, andsome together at different time points. For example, the cofactor,co-substrate, imine reductase, and substrate may be added first to thesolvent.

The solid reactants (e.g., enzyme, salts, etc.) may be provided to thereaction in a variety of different forms, including powder (e.g.,lyophilized, spray dried, and the like), solution, emulsion, suspension,and the like. The reactants can be readily lyophilized or spray driedusing methods and equipment that are known to those having ordinaryskill in the art. For example, the protein solution can be frozen at−80° C. in small aliquots, then added to a pre-chilled lyophilizationchamber, followed by the application of a vacuum.

For improved mixing efficiency when an aqueous co-solvent system isused, the imine reductase, and cofactor may be added and mixed into theaqueous phase first. The organic phase may then be added and mixed in,followed by addition of the imine reductase substrate and co-substrate.Alternatively, the imine reductase substrate may be premixed in theorganic phase, prior to addition to the aqueous phase.

The imine reductase reaction is generally allowed to proceed untilfurther conversion of substrates to product does not changesignificantly with reaction time, e.g., less than 10% of substratesbeing converted, or less than 5% of substrates being converted). In someembodiments, the reaction is allowed to proceed until there is completeor near complete conversion of substrates to product. Transformation ofsubstrates to product can be monitored using known methods by detectingsubstrate and/or product, with or without derivatization. Suitableanalytical methods include gas chromatography, HPLC, and the like.

In some embodiments of the process, the suitable reaction conditionscomprise a loading of substrates of at least about 5 g/L, 10 g/L, 20g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, or more, andwherein the method results in at least about 50%, 60%, 70%, 80%, 90%,95% or greater conversion of substrate compounds to product compound inabout 48 h or less, in about 36 h or less, or in about 24 h or less.

The engineered imine reductase polypeptides of the present disclosurewhen used in the process under suitable reaction conditions result in adiastereomeric excess of the desired secondary or tertiary amine productin at least 90%, 95%, 96%, 97%, 98%, 99%, or greater. In someembodiments, no detectable amount of the undesired diastereomericsecondary or tertiary amine product is formed.

In further embodiments of the processes for converting substratecompounds to amine product compound using the engineered imine reductasepolypeptides, the suitable reaction conditions can comprise initialsubstrate loadings to the reaction solution which is then contacted bythe polypeptide. This reaction solution is the further supplemented withadditional substrate compounds as a continuous or batchwise additionover time at a rate of at least about 1 g/L/h, at least about 2 g/L/h,at least about 4 g/L/h, at least about 6 g/L/h, or higher. Thus,according to these suitable reaction conditions, polypeptide is added toa solution having initial ketone and amine substrate loadings of each atleast about 20 g/L, 30 g/L, or 40 g/L. This addition of polypeptide isthen followed by continuous addition of further ketone and aminesubstrates to the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6g/L/h until a much higher final substrate loading of each at least about30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, 150 g/L, 200 g/L ormore, is reached. Accordingly, in some embodiments of the process, thesuitable reaction conditions comprise addition of the polypeptide to asolution having initial substrate loadings of each at least about 20g/L, 30 g/L, or 40 g/L followed by addition of further ketone and aminesubstrates to the solution at a rate of about 2 g/L/h, 4 g/L/h, or 6g/L/h until a final substrate loading of at least about 30 g/L, 40 g/L,50 g/L, 60 g/L, 70 g/L, 100 g/L or more, is reached. These substratesupplementation reaction conditions allow for higher substrate loadingsto be achieved while maintaining high rates of conversion of substratesto amine product of at least about 50%, 60%, 70%, 80%, 90% or greaterconversion.

In some embodiments of the processes, the reaction using an engineeredimine reductase polypeptide can comprise the following suitable reactionconditions: (a) substrate loading at about 5 g/L to 30 g/L; (b) about0.1 g/L to 10 g/L of the engineered polypeptide; (c) about 19 g/L (0.13M) to 57 g/L (0.39 M) of α-ketoglutarate; (d) about 14 g/L (0.08 M) to63 g/L (0.36 M) ascorbic acid; (e) about 1.5 g/L (3.8 mM) to 4.5 g/L(11.5 mM) of FeSO₄; (f) a pH of about 6 to 9; (g) temperature of about20° to 50° C.; and (h) reaction time of 2-24 hrs.

In some embodiments of the processes, the reaction using an engineeredimine reductase polypeptide can comprise the following suitable reactionconditions: (a) substrate loading at about 10 g/L to 100 g/L; (b) about1 g/L to about 50 g/L of engineered polypeptide; (c) NADH or NADPHloading at about 0.1 g/L to about 5 g/L; (d) pH of about 6 to 10; (g)temperature of about 20° to 50° C.; and (h) reaction time of 6 to 120hrs.

In some embodiments, additional reaction components or additionaltechniques carried out to supplement the reaction conditions. These caninclude taking measures to stabilize or prevent inactivation of theenzyme, reduce product inhibition, shift reaction equilibrium to amineproduct formation.

In further embodiments, any of the above described process for theconversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction; isolation;purification; and crystallization of product compound. Methods,techniques, and protocols for extracting, isolating, purifying, and/orcrystallizing the amine product from biocatalytic reaction mixturesproduced by the above disclosed methods are known to the ordinaryartisan and/or accessed through routine experimentation. Additionally,illustrative methods are provided in the Examples below.

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

7. EXAMPLES Example 1: Synthesis, Optimization, and Screening EngineeredPolypeptides Derived from CENDH Having Imine Reductase Activity

Gene Synthesis and Optimization:

The polynucleotide sequence encoding the reported wild-type opinedehydrogenase polypeptide CENDH from Arthrobacter Sp. Strain C1, asrepresented by SEQ ID NO: 2, was codon-optimized using the GeneIOSsynthesis platform (GeneOracle) and synthesized as the gene of SEQ IDNO: 1. The synthetic gene of SEQ ID NO: 1 was cloned into a pCK110900vector system (see e.g., US Patent Application Publication 20060195947,which is hereby incorporated by reference herein) and subsequentlyexpressed in E. coli W3110 fhuA. The E. coli W3110 expressed the opinedehydrogenase polypeptide CENDH under the control of the lac promoter.Based on sequence comparisons with other CENDH (and other amino aciddehydrogenases) and computer modeling of the CENDH structure docked tothe substrate, residue positions associated with the active site,peptide loops, solution/substrate interface, and potential stabilitypositions were identified. Briefly, directed evolution of the CENDH genewas carried out by constructing libraries of variant genes in whichthese positions associated with certain structural features weresubjected to mutagenesis. These libraries were then plated, grown-up,and screened using HTP assays as described in Examples 2 and 3 toprovide a first round (“Round 1”) of 41 engineered CENDH variantpolypeptides with imine reductase activity having even numbered sequenceidentifiers SEQ ID NO: 4-86. These amino acid differences identified inthese Round 1 variants were recombined to build new Round 2 librarieswhich were then screened for activity with the ketone substrate ofcompound (1b) and the amine substrate of compound (2b). This iminereductase activity screened for in Round 2 was not detectable in thenaturally occurring opine dehydrogenase CENDH polypeptide from which thevariants were derived. This second round of directed evolution resultedin the 7 engineered polypeptides having the even numbered sequenceidentifiers of SEQ ID NO: 88-100. These Round 2 variants of CENDH havefrom 4 to 10 amino acid differences relative to SEQ ID NO: 2 and havethe non-natural imine reductase activity of reductively aminatingcyclohexanone with butylamine to produce the secondary amine productcompound (2d).

Example 2: Production of Engineered Polypeptides Derived from CENDHHaving Imine Reductase Activity

The engineered imine reductase polypeptides were produced in E. coliW3110 under the control of the lac promoter. Enzyme preparations for HTPand SFP assays were made as follows.

High-Throughput (HTP) Growth, Expression, and Lysate Preparation.

Cells were picked and grown overnight in LB media containing 1% glucoseand 30 μg/mL chloramphenicol (CAM), 30° C., 200 rpm, 85% humidity. 20 μLof overnight growth were transferred to a deep well plate containing 380μL TB growth media containing 30 μg/mL CAM, 1 mM IPTG, and incubated for˜18 h at 30° C., 200 rpm, 85% humidity. Cell cultures were centrifugedat 4000 rpm, 4° C. for 10 min., and the media discarded. Cell pelletsthus obtained were stored at −80° C. and used to prepare lysate for HTPreactions as follows. Lysis buffer containing 1 g/L lysozyme and 1 g/LPMBS was prepared in 0.1 M phosphate buffer, pH 8.5 (or pH 10). Cellpellets in 96 well plates were lysed in 250 μL lysis buffer, withlow-speed shaking for 1.5 h on a titre-plate shaker at room temperature.The plates then were centrifuged at 4000 rpm for 10 mins at 4° C. andthe clear supernatant was used as the clear lysate in the HTP assayreaction.

Production of Shake Flask Powders (SFP):

A shake-flask procedure was used to generate engineered imine reductasepolypeptide powders used in secondary screening assays or in thebiocatalytic processes disclosed herein. Shake flask powder (SFP)provides a more purified preparation (e.g., up to 30% of total protein)of the engineered enzyme as compared to the cell lysate used in HTPassays and, among other things, allows for the use of more concentratedenzyme solutions. A single colony of E. coli containing a plasmidencoding an engineered polypeptide of interest is inoculated into 50 mLLuria Bertani broth containing 30 μg/ml chloramphenicol and 1% glucose.Cells are grown overnight (at least 16 hours) in an incubator at 30° C.with shaking at 250 rpm. The culture is diluted into 250 mL TerrificBroth (12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65mM potassium phosphate, pH 7.0, 1 mM MgSO₄) containing 30 μg/mlchloramphenicol, in a 1 L flask to an optical density of 600 nm (OD₆₀₀)of 0.2 and allowed to grow at 30° C. Expression of the imine reductasegene is induced by addition of isopropyl-β-D-thiogalactoside (“IPTG”) toa final concentration of 1 mM when the OD₆₀₀ of the culture is 0.6 to0.8. Incubation is then continued overnight (at least 16 hours). Cellsare harvested by centrifugation (5000 rpm, 15 min, 4° C.) and thesupernatant discarded. The cell pellet is resuspended with an equalvolume of cold (4° C.) 50 mM potassium phosphate buffer, pH 7.5, andharvested by centrifugation as above. The washed cells are resuspendedin two volumes of the cold 50 mM potassium phosphate buffer, pH 7.5 andpassed through a French Press twice at 12,000 psi while maintained at 4°C. Cell debris is removed by centrifugation (10,000 rpm, 45 minutes, 4°C.). The clear lysate supernatant is collected and stored at −20° C.Lyophilization of frozen clear lysate provides a dry shake-flask powderof crude engineered polypeptide. Alternatively, the cell pellet (beforeor after washing) can be stored at 4° C. or −80° C.

Example 3: HTP and SFP Screening of Engineered Polypeptides Derived fromCENDH for Improved Imine Reductase Activity with Various Ketone andAmine Substrate Compounds

HTP Screening Assays of Round 1 Engineered Polypeptides:

High-throughput screening used to guide primary selection of variantswas carried out in 96-well plates using clear cell lysate. The variantsfrom the first round of CENDH (SEQ ID NO: 2) mutagenesis were screenedusing two different HTP assay reactions as noted in Table 3A: (1) thecombination of ketone substrate compound (1a), pyruvate, and the aminesubstrate compound (2b), butylamine; and (2) the combination of ketonesubstrate (1b), cyclohexanone, and the amine substrate compound (2a),L-norvaline.

Secondary Screening of Round 1 Variants Using SFP Preparations:

SFP preparations were prepared for a selection of the Round 1 engineeredpolypeptides derived from CENDH exhibiting improved activity relative tothe CENDH wild-type of SEQ ID NO: 2 in one or both of the HTP screeningassays. These SFP preparations were submitted to a secondary screeningwith the three substrate combinations cyclohexanone/L-norvaline,cyclopentanone/L-norvaline, acetophenone/L-norvaline using the SFP assayas described in Table 3C.

HTP Screening Assays of Round 2 Engineered Polypeptides:

As described in Example 1, Round 2 libraries of engineered polypeptideswere prepared by recombining beneficial amino acid differencesidentified in Round 1 with a “backbone” polypeptide sequence of eitherSEQ ID NO: 6 or 86, which had the N198H, and N198E amino difference.These Round 2 libraries were subjected to HTP screening in thecyclohexanone/butylamine assay as described in Table 3B. Sevenengineered polypeptides were identified (SEQ ID NO: 88, 90, 92, 94, 96,98, and 100) having the imine reductase activity of convertingcyclohexanone and butylamine to the secondary amine product of compound(2d). This activity is not detectable in the naturally occurring opinedehydrogenase CENDH from which the polypeptides were derived. TheseRound 2 engineered polypeptides have from 4 to 10 amino acid differencesrelative to SEQ ID NO: 2.

Further Activity Screening of Round 2 Variants Using SFP Preparations:

SFP preparations were prepared for the seven Round 2 variants and thesepreparations were subjected several other activity assays using a rangeof ketone and amine substrate compounds as listed in Tables 3D and 3E.

Analysis of HTP and SFP Assay Reaction:

The combination method of Liquid Chromatography and Mass Spectrometry(LC-MS) was used as the primary analytical method to detect and quantifythe various HTP and SFP assay reaction results of Tables 3A, 3B, 3C, 3D,and 3E. Details of the LC-MS analysis are provided below.

LC-MS Analysis (Tables 3A-3E):

After the HTP assay or SFP assay reaction mixtures were shaken overnightat high-speed on a titre-plate shaker at room temperature, each reactionmixture was quenched with CH₃CN and diluted 10 fold in CH₃CN/H₂O/formicacid (50/50/0.1). The quenched and diluted reaction mixtures wereanalyzed by LC-MS in multiple reaction monitoring (MRM) mode. Therelevant LC instrumental parameters and conditions were as shown below.

LC Instrument Agilent HPLC 1200 series, API 3200 Qtrap Column PoroshellEC C18 50 × 3.0 mm, 2.7 μm, attached with Agilent C18 guard column(narrow bore) Mobile Phase Gradient (A: 0.5 mM perfluoroheptanoic acid(PFHA); B: MeCN) T (min) B % 0-1.5 3 9 30 12 30 13 3 20 3 Flow Rate 0.8mL/min Detection Q1MS, positive, DP 25 V, EP 10 V, CUR 30, IS 5000, TEM575° C., GS1 55, GS2 60. Column Temperature Not controlled InjectionVolume 2 μL Run time 20 min

The relevant MS parameters for the cyclohexanone/L-norvaline assayreaction were: [M+H]+: 200; Main fragment ions at CE=20 ev: 154, 118,83, 72, 55. The MRM transitions used for monitoring product formation:200/118; 200/72.

The relevant MS parameters for the pyruvate/butylamine assay reactionwere: [M+H]+: 146; Main fragment ion at CE=20 ev: 100. MRM transitionsused for monitoring product formation: 146/100.

The relevant MS parameters for the cyclohexanone/butylamine assayreaction were: [M+H]+: 156; Main fragment ions at CE=20 ev; 83, 74, 55.MRM transitions used for monitoring product formation: 156/83; 156/74;156/55.

The relevant MS parameters for the cyclopentanone/L-norvaline assayreaction: [M+H]⁺: 186; Main fragment ions at CE=20 ev; 140, 118, 79, 72.MRM transitions used for monitoring product formation: 186/72; 186/118;186/69.

The relevant MS parameters for the acetophenone/L-norvaline assayreaction: [M+H]⁺: 222; Main fragment ions at CE=20 ev; 118, 105, 72. MRMtransitions used for monitoring product formation: 222/118; 222/105;222/72.

The relevant MS parameters for the 2-methoxy cyclohexanone/butylamineassay reaction: [M+H]⁺: 186; Main fragment ions at CE=20 ev; 154, 113,98, 81. MRM transitions used for monitoring product formation: 186/154;186/81.

The relevant MS parameters for the cyclohexanone/methylamine assayreaction: [M+H]⁺: 114; Main fragment ions at CE=20 ev; 83, 55. MRMtransitions used for monitoring product formation: 114/83; 114/55.

The relevant MS parameters for the cyclohexanone/aniline assay reaction:[M+H]⁺: 176; Main fragment ions at CE=20 ev; 135, 94, 83, 55. MRMtransitions used for monitoring product formation: 176/94.

The relevant MS parameters for the 2-pentanone/butylamine assayreaction: [M+H]⁺: 144; Main fragment ions at CE=15 ev; 144, 114, 74, 71.MRM transitions used for monitoring product formation: 144/74; 144/71;144/43.

The relevant MS parameters for the hydroxy acetone/dimethylamine assayreaction: [M+H]⁺: 104; Main fragment ions at CE=25 ev; 86, 71, 59, 46,41. MRM transitions used for monitoring product formation: 104/86;104/46.

Example 4: HTP Screening of Engineered Polypeptides Derived from SEQ IDNO: 96 for Improved Stability and Imine Reductase Activity in PreparingCompounds (3n) and (3o)

The Round 2 engineered polypeptide having imine reductase activity ofSEQ ID NO: 96 was used to generate further engineered polypeptides ofTables 3F-3J which have further improved stability (e.g., activity at44° C.) and improved imine reductase activity (e.g., % conversion ofketone substrate compound (1j) to product). These engineeredpolypeptides, which have the amino acid sequences of even-numberedsequence identifiers SEQ ID NO: 112-750, were generated from the“backbone” amino acid sequence of SEQ ID NO: 96 using the directedevolution methods of Examples 1 and 2 together with HTP assay methods asnoted in Tables 3F-3J. Further details of amine product LC-MS analysisof the assay mixtures is provided below.

LC-MS Analysis for Amine Product Compound (3n):

After the HTP assay mixtures were shaken overnight at 250 rpm on atitre-plate shaker at 35° C., each reaction mixture was quenched byadding 250 μL CH₃CN, shaken, and centrifuged at 4000 rpm and 4° C. for10 min. 20 μL of the quenched mixture was diluted 10 fold in 180 μLCH₃CN/H₂O (50/50) with mixing. 10 μL of this 10-fold dilution mixturewas then further diluted in 190 μL CH₃CN/H₂O (50/50) for a total 400fold diluted mixtures. These mixtures were analyzed by LC-MS in MRMmode. Formation of the product compound (1i),N-butyl-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-amine, using the MRMtransition: 234/161. Additional relevant LC-MS instrumental parametersand conditions were as shown below.

Instrument Agilent HPLC 1200 series, API 3200 Qtrap Column Poroshell 120EC C18 50 × 3.0 mm, 2.7 μm Mobile Phase Gradient (A: 0.1% formic acid inwater; B: MeCN; A:B = 36:64) Flow Rate 0.8 mL/min Run time 0.7 min PeakRetention Times Compound (3n): 0.55 min Column Temperature 25° C.Injection Volume 2 μL MS Detection Qtrap3200; MRM234/161 (forN-butyl-5-methoxy-1,2,3,4- tetrahydronaphthalen-2-amine); 0-0.4 minbypass MS MS Conditions MODE: MRM; CUR: 30; IS: 5500; CAD: medium; TEM:560° C.; GS1: 60; GS2: 60; DP: 30; EP: 9; CE: 25; CXP: 3; DT: 380 ms

LC-MS Analysis for Amine Product Compound (3o):

After the HTP assay mixtures were shaken overnight at 250 rpm on atitre-plate shaker at 35° C., each reaction mixture was quenched with100 μL CH₃CN, heat-sealed, shaken, and centrifuged at 4000 rpm, 4° C.,for 10 min. 20 μL of the quenched mixture was diluted 10-fold in 180 μLCH₃CN/H₂O (50/50) with mixing. The 10-fold diluted reaction mixtureswere analyzed by LC-MS in multiple reaction monitoring (MRM) mode. Therelevant instrumental parameters and conditions were as shown below.

Instrument Agilent HPLC 1260 coupled with API 2000 Qtrap ColumnPoroshell 120 EC C18 50 × 3.0 mm, 2.7 μm (Agilent Technologies, SantaClara, CA) Mobile Phase Gradient (A: 0.1% formic acid in water; B: MeCN)Flow Rate 0.8 mL/min T (min) B % 0-0.8 25 2-2.5 90 2.6-3.5   30 Run time3.5 min Peak Retention Time Compound (3o): 2.18 min Column Temperature25° C. Injection Volume 10 μL MS Detection Compound (3o) detected inQtrap2000 MRM mode: parent ion at m/z 336.25, fragment ion at m/z 154 MSConditions CUR: 30; IS: 4500; CAD: 6; TEM: 550° C.; GS1: 60; GS2: 60;DP: 31; EP: 10; CE: 30; CXP: 3

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An engineered polynucleotide encoding anengineered polypeptide having imine reductase activity, wherein saidpolypeptide has at least 90% sequence identity to the polypeptide SEQ IDNO:2, and comprises a substitution at a position corresponding toposition 293 of SEQ ID NO:2, wherein the amino acid at position 293 hasbeen replaced with an aromatic, non-polar, aliphatic, basic, or polarresidue.
 2. The engineered polynucleotide encoding the engineeredpolypeptide having imine reductase activity of claim 1, wherein saidpolypeptide has at least 90% sequence identity to the polypeptide of SEQID NO:2, wherein the amino acid at the position corresponding toposition 293 of SEQ ID NO:2 has been replaced with leucine.
 3. Theengineered polynucleotide encoding the engineered polypeptide of claim1, wherein said polypeptide is capable of converting substrate compound(1a) pyruvate,

and substrate compound (2b) butylamine

to product compound (3b), N-2-(butylamino)propanoic acid,

under suitable reaction conditions.
 4. The engineered polynucleotideencoding the engineered polypeptide of claim 1, wherein said polypeptideis capable of converting substrate compound (1b) cyclohexanone,

and substrate compound (2a) L-norvaline

to product compound (3c), (S)-2-(cyclohexylamino)pentanoic acid,

under suitable reaction conditions.
 5. The engineered polynucleotideencoding the engineered polypeptide of claim 1, wherein said polypeptideis capable of converting substrate compound (1b) cyclohexanone,

and substrate compound (2b) butylamine

to product compound (3d), N-butylcyclohexanamine,

under suitable reaction conditions.
 6. The engineered polynucleotideencoding the engineered polypeptide of claim 1, wherein said polypeptideis capable of converting substrate compound (1i),

and substrate compound (2b)

to product compound (3n),

under suitable reaction conditions.
 7. The engineered polynucleotideencoding the engineered polypeptide of claim 1, wherein said polypeptideis capable of converting substrate compound (1j),

and substrate compound (2b)

to product compound (3o),

under suitable reaction conditions.
 8. A process for preparing an aminecompound of formula (III),

wherein (a) R¹ and R² are independently selected from the groupconsisting of a hydrogen atom, an optionally substituted alkyl, anoptionally substituted alkenyl, an optionally substituted alkynyl, anoptionally substituted alkoxy, an optionally substituted carboxy, anoptionally substituted aminocarbonyl, an optionally substitutedheteroalkyl, an optionally substituted heteroalkenyl, an optionallysubstituted heteroalkynyl, an optionally substituted carboxyalkyl, anoptionally substituted aminoalkyl, an optionally substituted haloalkyl,an optionally substituted alkylthioalkyl, an optionally substitutedcycloalkyl, an optionally substituted aryl, an optionally substitutedarylalkyl, an optionally substituted heterocycloalkyl, an optionallysubstituted heteroaryl, and an optionally substituted heteroarylalkyl;(b) R¹ and R² are linked to form a 3-membered to 10-membered ring; (c)R³ and R⁴ groups are independently selected from a hydrogen atom, andoptionally substituted alkyl, an optionally substituted alkenyl, anoptionally substituted alkynyl, an optionally substituted alkoxy, anoptionally substituted carboxy, an optionally substituted aminocarbonylan optionally substituted heteroalkyl, an optionally substitutedheteroalkenyl, an optionally substituted heteroalkynyl, an optionallysubstituted carboxyalkyl, an optionally substituted aminoalkyl, anoptionally substituted haloalkyl, an optionally substitutedalkylthioalkyl, an optionally substituted cycloalkyl, an optionallysubstituted aryl, an optionally substituted arylalkyl, an optionallysubstituted heterocycloalkyl, an optionally substituted heteroaryl, andan optionally substituted heteroarylalkyl, with the proviso that both R³and R⁴ cannot be hydrogen; (d) R³ and R⁴ are linked to form a 3-memberedto 10-membered ring; or (f) at least one of R¹ and R² is linked to atleast one of R³ and R⁴; wherein optionally, the carbon atom and/or thenitrogen indicated by * is chiral; and the process comprising contactinga compound of formula (I),

wherein R¹, and R² are as defined above; and a compound of formula (II),

wherein R³, and R⁴ are as defined above; with an engineered polypeptidehaving imine reductase activity in presence of a cofactor under suitablereaction conditions, wherein the engineered polypeptide is encoded bythe engineered polynucleotide of claim
 1. 9. The process of claim 8, inwhich R³ and R⁴ are linked to form a 3-membered to 10-membered ring. 10.The process of claim 8, in which the substrate compound of formula (II)is selected from methylamine, dimethylamine, isopropylamine, butylamine,isobutylaminel, L-norvaline, aniline, (S)-2-aminopent-4-enoic acid,pyrrolidine, and hydroxypyrrolidine.
 11. The process of claim 8, inwhich at least one of R¹ and R² of the compound of formula (I) is linkedto at least one of R³ and R⁴ of the amine compound of formula (II),whereby the process for preparing the amine compound of formula (III)comprises an intramolecular reaction.
 12. The process of claim 8, inwhich the suitable reaction conditions comprise: (a) substrate loadingat about 10 g/L to 100 g/L; (b) about 0.1 g/L to about 50 g/L of theengineered polypeptide; (c) about 0.05 g/L (0.001 M) to about 2.5 g/L(0.050 M) of NAD(P)H; (d) a pH of about 6 to 10; (e) temperature ofabout 20° to 50° C.; and (f) reaction time of 2-120 hrs.
 13. A vectorcomprising the engineered polynucleotide of claim
 1. 14. The vector ofclaim 13, further comprising at least one control sequence.
 15. A hostcell comprising at least one vector of claim
 13. 16. A host cellcomprising at least one vector of claim 14.